Small molecule conjugates for intracellular delivery of biologically active compounds

ABSTRACT

The invention provides novel compounds and conjugates of these compounds useful for the delivery of biologically active substances. Further novel design criteria for chemically stabilized siRNA particular useful when covalently attached to a delivery polymer to achieve in vivo mRNA knock down are disclosed therein.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2011/073718, filed Dec. 22, 2011, which claims prioritybenefit to U.S. Provisional Patent Application No. 61/427,845 filed Dec.29, 2010, the content of each of which is hereby incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Aug. 26, 2013, is named P5647R1_SL.txt and is 449,820bytes in size.

FIELD OF THE INVENTION

The present invention relates to novel small molecule conjugates usefulfor the delivery of biologically active substances, such as nucleicacids, peptides and proteins. The delivery of nucleic acids and othersubstantially cell membrane impermeable compounds into a living cell ishighly restricted by the complex membrane system of the cell.

One means that has been used to deliver a biologically active substancesuch as nucleic acids in vivo has been to attach the biologically activesubstance to either a small targeting molecule or a hydrophobic moleculesuch as a lipid or sterol. While some delivery and activity has beenobserved with these conjugates, the biologically active substance doserequired with these methods has been prohibitively large, resultingoften in undesired toxicity effects in vivo. Provided herein are smallmolecule compounds that can be conjugated to a biologically activesubstance and mediate successful delivery of said biologically activesubstance into a cell. Surprisingly it has been found that significantlydecreased doses of the biologically active substance are now sufficientfor successful delivery when using the novel compounds provided herein.Thus, the novel compounds provide a powerful tool for the delivery ofbiologically active substances with considerably limited toxicity invivo.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to the compounds offormula

wherein

-   -   Y is a linker group selected from —(CH₂)₃— or        —C(O)—N—(CH₂—CH₂—O)_(p)—CH₂—CH₂—;    -   R¹ is —(C1-6) alkyl;        -   —(CH₂)-naphthyl; or        -   —(CH₂)_(m)-phenyl, which phenyl is unsubstituted or up to            four times substituted with a substituent independently            selected from            -   —NO₂,            -   —CN,            -   Halogen,            -   —O—(CH₂)-phenyl,            -   —O—(C1-6) alkyl, or            -   —C(O)—NH₂;    -   R² is hydrogen;        -   —(CH₂)_(k)—N—C(Ph)₃, which phenyl rings are unsubstituted or            independently substituted with —O—(C1-4)alkyl;        -   —(CH₂)_(k)—C(O)—NH₂;        -   —(CH₂)_(k)-phenyl;        -   —(C1-6) alkyl, which is unsubstituted or once substituted            with —S—CH₃;    -   R³ is —NH-phenyl, which phenyl group is further substituted with        a substituent independently selected from        -   —(CH₂)—OH; or        -   —(CH₂)—O—C(O)—O-(4-nitro-phenyl);    -   k is 1, 2, 3, 4, 5, 6;    -   m is 1, 2, 3 or 4;    -   n is 0 or 1; and    -   p is an integer from 1 to 20.

In another embodiment, the compounds of formula (I) may have thespecific conformation as shown in formula (Ia),

wherein all substituents R¹, R², R³ and Y as well as the variables k, m,n, and p have the meaning given above.

In yet another embodiment, the present invention is directed tocompounds of formula (I) or (Ia), wherein Y is —(CH₂)₃—; and allremaining substituent groups have the meaning given above.

In yet another embodiment, the present invention is directed tocompounds of formula (I) or (Ia), wherein Y is—C(O)—N—(CH₂—CH₂—O)_(p)—CH₂—CH₂—; and all substituent groups have themeaning given above.

In yet another embodiment, there are provided the compounds of formulae(I) or (Ia), wherein

-   -   Y is —(CH₂)₃—;    -   R² is —(CH₂)_(k)—N—C(Ph)₃, which phenyl rings are unsubstituted        or independently substituted with —O—(C1-4)alkyl; and    -   R³ is —NH-phenyl, which phenyl group is further substituted with        -   —(CH₂)—O—C(O)—O-(4-nitro-phenyl);    -   n is 0; and    -   R¹ and k have the meanings given above.

In yet another embodiment, there are provided the compounds of formulae(I) or (Ia), wherein

-   Y is —C(O)—N—(CH₂—CH₂—O)_(p)—CH₂—CH₂—;-   R² is —(CH₂)_(k)—N—C(Ph)₃, which phenyl rings are unsubstituted or    independently substituted with —O—(C1-4)alkyl; and    -   R³ is —NH-phenyl, which phenyl group is further substituted with        —(CH₂)—O—C(O)—O-(4-nitro-phenyl);-   n is 0; and-   R¹, k and p have the meanings given above.

DETAILED DESCRIPTION OF THE INVENTION

The term “(C1-6) alkyl” as used herein means a linear or branched,saturated hydrocarbon containing from 1 to 6 carbon atoms. PreferredC1-6 alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl,2-butyl and the like.

The term “halogen” as used herein means fluorine, chlorine, bromine, oriodine with fluorine and chlorine being preferred.

The compounds according to the present invention can be generallyobtained using methods known to the person of ordinary skill in the artof organic- or medicinal chemistry. More particularly the compounds offormula (Ia), wherein Y is —(CH₂)₃— and n=0, can be obtained usingcompound (A) as starting material.

The synthesis of (A) is described inter alia in WO2001/070415.

The compound of formula (A) is further reacted in the presence ofHuenig's base and ethyl acetate (AcOET), followed by the addition ofdihydrofuran-2,5-dione in THF, to give the compounds of formula (B)

The compounds of formula (B) are further reacted with an amine offormula (C),

to give the compounds of formula (Ia).

The compounds of formula (I) or (Ia) are useful as ligands onbiologically active substances, such as nucleic acids, peptides orproteins, to which they are covalently attached. Preferably, thecovalent bond is created by the reaction of a suitable functional group,such as i.e. a primary amine group, in the biologically active substancewith the activated carbonyl group in the —O—C(O)—O— moiety of R³ asdefined herein before. Hence provided herein is a conjugate comprisingthe compounds of formula (I) or (Ia) and a biologically activesubstance.

The term “biologically active substance” as used herein refers to aninorganic or organic molecule including a small molecule, peptide (e.g.cell penetrating peptides), protein, carbohydrate (includingmonosaccharides, oligosaccharides, and polysaccharides), nucleoprotein,mucoprotein, lipoprotein, synthetic polypeptide or protein, or a smallmolecule linked to a protein, glycoprotein, steroid, nucleic acid (anyform of DNA, including cDNA, or RNA, or a fragment thereof), nucleotide,nucleoside, oligonucleotides (including antisense oligonucleotides, LNAand siRNA), gene, lipid, hormone, or combination thereof, that causes abiological effect when administered in vivo to an animal, including butnot limited to birds and mammals, including humans. Preferably, saidbiologically active substance is a peptide or a nucleic acid. Preferrednucleic acids used herein are siRNAs.

The conjugate comprising the present compounds covalently attached to abiologically active substance shows an improved ability to be taken upby cells compared to said biologically active substance alone. Once theconjugate is delivered into the cell and trafficking to the lysosome,the corresponding biologically active substance is released by enzymaticcleavage. This cleavage preferably takes place when a di-peptide motif,preferably consisting of the sequence α- or β-(phenyl)alanine and lysineas present in the compounds of formula (I) or (Ia) is incorporated inthe conjugate (see scheme 1). Most preferably the conjugate contains thedi-peptide motif and a spacer such as the p-aminobenzylcarbamate spacer(Bioconjugate Chem. 2002, 13, 855) that spontaneously fragments once theamide bond C-terminal of the di-peptide motif is cleaved as exemplifiedfor siRNAs in scheme 2. Hence the conjugates comprising compounds offormula (I) or (Ia) are also referred to as dipeptide containingcholesterol conjugates. Enzymatic cleavage of the biologically activesubstance from the dipeptide containing cholesterol conjugates of thisinvention is catalyzed by innate proteases of the cell. One example ofan innate protease capable of cleaving the di-peptide motif present inthe compounds of formula (I) or (Ia) is Cathepsin B. Cathepsin B is aknown ubiquitous cysteine protease located in the lysosomes of mammaliancells (Bioconjugate Chem. 2002, 13, 855; J. Med. Chem. 2005, 48, 1344;Nat. Biotechnology 2003, 21, 778). Thus, the di-peptide motif describedabove is also referred to as Cathepsin-cleavable dipeptide-motif.

The present invention therefore also provides a method for delivery of abiologically active substance, into cells wherein said biologicallyactive substance may subsequently be cleaved off the conjugate to unfolda therapeutic activity.

In a further embodiment of the present invention, there is provided aconjugate of the compounds of formula (I) or (Ia) covalently attached toa biologically active compound, preferably siRNA or a peptide moiety.Preferably said peptide moiety is a peptide that exhibits membraneperturbing properties like cell penetrating peptides or amphiphilicpeptides.

Conjugates of formula (I) or (Ia) covalently attached to a biologicallyactive substance are designated herein as formula (II) or (IIa),respectively.

Therefore, in a further embodiment, the present invention provides acompound of formula

whereinR^(a) is —(CH₂)_(k)—NH₂;R¹ and k have the meanings given for formula (I) above; andthe biologically active substance is a nucleic acid, a protein or apeptide.

In a more specific embodiment, the present invention provides compoundsof formula

whereinR^(a) is —(CH₂)_(k)—NH₂;R¹ and k have the meanings given for formula (I) above; andthe biologically active substance is a nucleic acid, a protein or apeptide.

In a preferred embodiment, the biologically active substance in formula(II) or (IIa) is a nucleic acid, most preferably a siRNA.

In another preferred embodiment, the biologically active substance informula (II) or (IIa) is a protein or a peptide.

The compounds of formula (II) or (IIa) may have valuable properties intherapy. Therefore, in a further embodiment, there are provided thecompounds of formula (II) or (IIa) for use as medicaments.

Another embodiment of the invention is a pharmaceutical compositioncomprising the conjugates of the compounds of formula (I) or (Ia)covalently attached to a biologically active substance.

In still another embodiment of the invention there is provided apharmaceutical composition comprising the compounds of formula (IIa)together with pharmaceutically acceptable excipients.

Below embodiments are exemplified for conjugates of the compounds offormula (I) or (Ia) covalently attached to siRNA, thus the compounds offormula (II) or (IIa) wherein the biological active substance is siRNA.It is understood that these embodiments are also applicable for otherbiologically active substances such as peptides and proteins.

The covalent attachment of the siRNA to the compounds of formula (I) or(Ia) is achieved via reaction of a suitable nucleophilic group, i.e. aprimary amine group, in the siRNA with the activated —C(O)— group in R³of said compounds of formula (I) or (Ia). The activation of that —C(O)—group is obtained by a p-nitrophenoxy carbonate as shown in scheme 2below.

The p-nitrophenyl activated carbonate may for example be reacted withthe siRNA equipped with a hexylamino-linker to generate a carbamatelinkage to yield the siRNA conjugate. Once the siRNA is taken upintracellularly and transfected to the lysosome the compounds of formula(II) or (IIa) wherein the biological active substance is siRNA arecleaved by the protease activity releasing the siRNA as also shown inscheme 2. The cholesterol moiety of the conjugate of the compounds offormula (II) or (IIa) modifies the PK properties of siRNA in such a waythat systemic administration enables gene silencing in vivo.

In one embodiment the compounds of formula (II) or (IIa) wherein thebiological active substance is siRNA is co-administered with a deliverypolymer. Delivery polymers provide a means of disrupting cell membranesand mediate endosomal release. In another embodiment, said deliverypolymer and the siRNA conjugate of the invention are not covalentlyattached and synthesized separately and may be supplied in separatecontainers or a single container. Delivery polymers for oligonucleotidessuch as siRNA are well known in the art. For example, Rozema et al., inU.S. Patent Publication 20040162260 demonstrated a means to reversiblyregulate membrane disruptive activity of a membrane active polyamine.Reversible regulation provided a means to limit activity to theendosomes of target cells, thus limiting toxicity. Their method reliedon reaction of amines on the polyamine with 2-propionic-3-methylmaleicanhydride. This modification converted the polycation to a polyanion viaconversion of primary amines to carboxyl-containing groups andreversibly inhibited membrane activity of the polyamine. To enableco-delivery of the nucleic acid with the delivery vehicle, the nucleicacid was covalently linked to the delivery polymer. In U.S. provisionalpatent application 61/307,490 a new generation of delivery polymers isdescribed. Therein, membrane active polyamine comprising an amphipathicterpolymer formed by random polymerization of amine-containing monomers,lower hydrophobic monomers, and higher hydrophobic monomers areprovided. This new generation of delivery polymers removed therequirement that polynucleotide and polymer are associated either bycovalent linkage or by charge-charge interaction.

Non-limiting examples of delivery polymers used for co-administrationwith the siRNA conjugates of the invention are membrane activepolyamines and poly(vinyl ether) (PBAVE), Dynamic PolyConjugates (DPC;Rozema et al. 2007) and improved DPCs as disclosed in U.S. provisionalpatent application 61/307,490.

In a further embodiment, a new chemical siRNA modification pattern forfunctional in vivo delivery is provided. This new chemical siRNAmodification pattern is especially useful with delivery vehicles whichdisplay a relatively strong endosomal/lysosomal retention.

It was found that siRNA stabilization against degradation byendosomal/lysosomal-localized nucleases such as DNAse II stronglyimproves target knock down. Such stabilization may directly effect theamount of siRNA released into the cytoplasm where the cellular RNAimachinery is located. Only the siRNA portion available in the cytoplasmwill trigger the RNAi effect.

In addition to poor pharmacokinetic characteristics, siRNAs aresusceptible to nucleases in the biological environment when administeredas such into the circulation without a protecting delivery vehicle.Accordingly, many siRNAs are rapidly degraded either extracellularly inthe tissue and blood stream or after intracellular uptake (endosome).

One well known nuclease localized in the endosomal/lysosomal compartmentis DNase II. This enzyme is active at a pH below 6-6.5 with maximumactivity in the pH-range of 4.5-5, reflecting conditions present in theacidified environment of the endosomal/lysosomal compartment. Thefollowing RNA degradation pathways induced by DNase II were identifiedin vitro and are disclosed in this invention:

A. RNA strands containing at least one 2′-OH nucleotide are rapidlydegraded via a cyclic pentavalent phosphorus intermediate, leading to2′-3′ cyclic phosphates at the 5′-cleavage product. The formation of thepentavalent intermediate can be inhibited by nucleotides lacking a 2′-OHgroup such as 2′-deoxy, 2′-O-methyl (2′-OMe) or 2′-deoxy-2′-fluoro(2′-F) nucleotides.B. Additionally, RNA is degraded in a 5′-exonucleolytic pathwayindependent of the 2′-modification on the 5′-terminal nucleotides. Thisdegradation pathway can be inhibited by 5′-terminal non-nucleotidemoieties, like e.g. cholesterol, aminoalkyl-linker or a phosphothioateat the first internucleotide linkage.C. A 5′-phosphate also protects and slows down the exonucleolyticcleavage kinetics, but can not fully block this pathway. This is mostprobably due to the cleavage of the 5′-phosphate by phosphatases or aninherent phosphatase activity of the DNase II enzyme preparation used inthe stability assay.D. The best protection was achieved with oligonucleotides lacking any2′-OH nucleotide within the strand, starting with a 2′-OMe nucleotide atthe 5′-end connected by a phosphorothioate (PTO) linkage to the secondnucleotide. Other terminal non-2′-OH nucleotides also protect againstthe 5′-exo degradation, but to a lower extent compared to the 2′-OMemodification.

Hence the inventors of the present invention found that siRNAs can besignificantly stabilized when using the following design, wherein anoligonucleotide is provided with an antisense strand with themodification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strandwith the modification pattern 5′-(Z3)n_(s)-3′, wherein

w is independently a 5′-phosphate or 5′-phosphothioate or H,

Z1 is independently a 2′-modified nucleoside.

Z2 is independently a 2′-deoxy nucleoside or 2′-Fluoro-modifiednucleoside,

Z3 is independently a 2′-modified nucleoside,

n_(a) is 8-23 and n_(s) is 8-25.

In one preferred embodiment an oligonucleotide is provided with anantisense strand with the modification pattern:5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strand with the modificationpattern 5′-(Z3)n_(s)-3′, wherein Z1 is a 2′-Fluoro-modified nucleosideor a 2deoxy-nucleoside and all remaining substituents as well as thevariables n_(a) and n_(s) have the meaning given above.

In one preferred embodiment an oligonucleotide is provided with anantisense strand with the modification pattern:5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strand with the modificationpattern 5′-(Z3)n_(s)-3′, wherein Z3 is a 2′-O-Methyl modifiednucleoside, a 2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside andall remaining substituents as well as the variables n_(a) and n_(s) havethe meaning given above.

In one preferred embodiment an oligonucleotide is provided with anantisense strand with the modification pattern:5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strand with the modificationpattern 5′-(Z3)n_(s)-3′, wherein Z1 is a 2′-Fluoro-modified nucleosideor a 2deoxy-nucleoside and Z3 is a 2′-O-Methyl modified nucleoside, a2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside and all remainingsubstituents as well as the variables n_(a) and n_(s) have the meaninggiven above.

The nucleosides in the nucleic acid sequence of the oligonucleotide withthe novel modification pattern can either be linked by 5′-3′phosphodiesters or 5′-3′ phosphorothioates.

As used herein, the “anti-sense” strand is the siRNA strand that iscomplementary to the target mRNA and that will be binding to the mRNAonce the siRNA is unwound.

The sense strand of said siRNA comprising the novel modification patternis complimentary to the antisense strand.

Said siRNA comprising the novel modification pattern proofed to beparticularly advantageous when covalently attached to a delivery polymeras exemplified by Rozema et al. (Dynamic PolyConjugates (DPC; Rozema etal. 2007). Potency and duration of effect can be significantly enhancedemploying the siRNA modification strategy outlined in this invention.

In another embodiment, said siRNA comprising the novel modificationpattern are especially useful when conjugated to small molecules thatalter the pharmacokinetic properties of siRNA such as cholesterol or thecompounds of formula (I) and (Ia) provided herein. In one embodiment aconjugate of a small molecule and an oligonucleotide is provided whereinthe oligonucleotide has the following modification pattern: theantisense strand with the modification pattern:5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strand with the modificationpattern 5′-(Z3)n_(s)-, wherein the substituents as well as the variablesn_(a) and n_(s) have the meaning given above. In one embodiment saidsmall molecule is cholesterol. In another embodiment said small moleculeis a compound of formula (I) or (I a), resulting in compounds of formula(II) or (IIa).

Preferably, said siRNAs conjugates are co-administered with a deliverypolymer. Suitable delivery polymers are described above.

In one embodiment, said siRNA comprising the novel modification patternare especially useful when conjugated to a ligand that is known to bindto a specific receptor which internalizes the conjugate into a cell.Particularly, the asialoglycoprotein receptor (ASGPR) expressed onhepatocytes is a well-known receptor enabling the clearance (endocytosisand lysosomal degradation) of desialylated proteins from circulation. Ithas been shown that the N-Acetyl-D-galactosamine has a high bindingaffinity for the receptor, especially when presented multivalent andwhen the galactose residues are properly spaced (J Biol Chem, 2001, 276,37577). In order to utilize this high capacity receptor for receptormediated endocytosis of the biologically active substance, the syntheticligand shown below was prepared to be covalently attached to the siRNAscomprising the novel modification pattern. Since this type ofendocytosis leads to lysosomal degradation of the internalized materialthe siRNA must be prepared in such a way that it is stable in thelysosome, which is now solved by the novel modification pattern outlinedabove.

The ligand for the ASGPR is attached via an amide bond to thebiologically active substance. The amide bond formation can beestablished with the aid of NHS chemistry. The ligand employed in theconjugation reaction is shown below (formula III). For interaction withthe ASGPR the O-acetate groups need to be removed as shown in (formulaIV) for an siRNA.

In one embodiment of the invention, a conjugate of a compound of formulaIV and an oligonucleotide is provided, wherein the oligonucleotide hasthe following modification pattern: the antisense strand with themodification pattern 5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strandwith the modification pattern 5′-(Z3)n_(s)-, wherein the substituents aswell as the variables n_(a) and n_(s) have the meaning given above. Saidconjugate is also referred to as GalNAc palmitoyl conjugate. Preferably,said GalNAc palmitoyl conjugate are co-administered with a deliverypolymer. Suitable delivery polymers are described above.

It was found that for these modification patterns cleavable linkersproofed to be advantageous compared to stably linked small moleculeligands. Possible cleavable linkers are a di-peptide motif asexemplified in scheme 1 or a cleavable RNA-linker comprising 2′-OHcontaining nucleotides. The cleavable RNA-linker is especially useful inconnection with the siRNAs having the novel modification pattern (fully2′-modified siRNA) described above.

In principle a nuclease cleavage site can be introduced by 3′- or5′-overhangs containing at least one 2′-OH nucleotide at either thesense or the antisense strand. The final active siRNA species isgenerated by intracellular nuclease processing. Also, the use of definedcleavage sites implemented by 2′-OH nucleotides within the base pairedregion is possible. This can be done using at least one 2′-OH nucleotidecomplementary to the opposite strand or by introduction of either atleast one mismatched 2′-OH nucleotide or a hairpin/bulge containing atleast one 2′-OH nucleotide.

In contrast to other cleavable linker chemistries the use of definedcleavage sites by introduction of 2′-OH nucleotides lead to a moreversatile conjugation approach. By introducing selective cleavage siteson one or on both strands of the siRNA either at the 3′ and/or the5′-end or within the duplex structure, multiple conjugation is possible.

Accordingly, in one embodiment, a conjugate of a small molecule and anoligonucleotide is provided wherein

-   -   a) the small molecule comprises a nucleotide linker comprising        1-10 preferably 1-5, most preferably 1-3 2′OH-nucleotides;    -   b) the oligonucleotide has the following modification pattern:        the antisense strand with the modification pattern        5′-(w)-(Z1)-(Z2)-(Z3)n_(a)-3′ and a sense strand with the        modification pattern 5′-(Z3)n_(s)-, wherein the substituents as        well as the variables n_(a) and n_(s) have the meaning given        above; and    -   c) the oligonucleotide is covalently attached to the nucleotide        linker of the small molecule.

The nucleotide linker is cleaved by intracellular nucleases such asDNAse II after internalization of the conjugate into the endosome, thusreleasing the siRNA.

Preferably, said conjugate is co-administered with a delivery polymer.Suitable delivery polymers are described above.

In another embodiment of the invention a compound of formula (V) isprovided. This compound comprises a cholesterol moiety, and a nucleotidelinker comprising 1-10 preferably 1-5, most preferably 1-32′OH-nucleotides. This nucleotide linker is useful for covalentlyattaching an oligonucleotide such as a siRNA to the compound of formula(V). Preferably, said oligonucleotide has the novel modification patternoutlined above. Hence in another embodiment a conjugate of a compound offormula (V) and an oligonucleotide is provided, wherein theoligonucleotide is covalently attached to the nucleotide linker of thecompound of formula (V).

The nucleotide linker is cleaved by intracellular nucleases such asDNAse II after internalization of the conjugate of a compound of formula(V) and an oligonucleotide into the endosome, thus releasing the siRNA.

Preferably, said conjugate of a compound of formula (V) and anoligonucleotide is co-administered with a delivery polymer. Suitabledelivery polymers are described above.

In another embodiment, said delivery polymer and the conjugate of acompound of formula (V) and an oligonucleotide of the invention are notcovalently attached and synthesized separately and may be supplied inseparate containers or a single container.

DEFINITIONS

The term “small molecule” as used herein, refers to organic or inorganicmolecules either synthesized or found in nature, generally having amolecular weight less than 10,000 grams per mole, optionally less than5,000 grams per mole, and optionally less than 2,000 grams per mole.

The term “peptide” as used herein refers to any polymer compoundproduced by amide bond formation between an .alpha.-carboxyl group ofone D- or L-amino acid and an .alpha.-amino group of another D- orL-amino acid. The term “protein” as used herein refers to polypeptidesof specific sequence of more than about 50 residues.

The term “di-peptide motif” as used herein refers to any motifcomprising an amide bond formed by either the D- or L-alpha or betaamino group of a first amino acid with the alpha-carboxyl group of asecond D- or L-amino acid.

As used herein, the term “amino acid” refers to any molecule thatcontains both amine and carboxyl functional groups. Thus the term “aminoacid” refers to both natural, non-natural and synthetic amino acids. Anynatural amino acids used in the present invention are referred to hereinby their common abbreviations.

The term “ligand” as used herein refers to a moiety that is capable ofcovalently or otherwise chemically binding a biologically activesubstance. The term “ligand” in the context of the invention ispreferably a compound of formula (I) or (Ia) covalently attached to abiologically active substance.

The term “biologically active substance” as used herein refers to aninorganic or organic molecule including a small molecule, peptide (e.g.cell penetrating peptides), protein, carbohydrate (includingmonosaccharides, oligosaccharides, and polysaccharides), nucleoprotein,mucoprotein, lipoprotein, synthetic polypeptide or protein, or a smallmolecule linked to a protein, glycoprotein, steroid, nucleic acid (anyform of DNA, including cDNA, or RNA, or a fragment thereof), nucleotide,nucleoside, oligonucleotides (including antisense oligonucleotides, LNAand siRNA), gene, lipid, hormone, or combination thereof, that causes abiological effect when administered in vivo to an animal, including butnot limited to birds and mammals, including humans. Preferably, saidbiologically active substance is a peptide or a nucleic acid. Preferrednucleic acids used herein are siRNAs.

The term “nucleic acid” as used herein means an oligomer or polymercomposed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides,or compounds produced synthetically (e.g., PNA as described in U.S. Pat.No. 5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. Non-naturallyoccurring nucleic acids are oligomers or polymers which containnucleobase sequences which do not occur in nature, or species whichcontain functional equivalents of naturally occurring nucleobases,sugars, or inter-sugar linkages, like peptide nucleic acids (PNA),threose nucleic acids (TNA), locked nucleic acids (LNA), or glycerolnucleic acids (GNA). This term includes oligomers that contain thenaturally occurring nucleic acid nucleobases adenine (A), guanine (G),thymine (T), cytosine (C) and uracil (U), as well as oligomers thatcontain base analogs or modified nucleobases. Nucleic acids can derivefrom a variety of natural sources such as viral, bacterial andeukaryotic DNAs and RNAs. Other nucleic acids can be derived fromsynthetic sources, and include any of the multiple oligonucleotides thatare being manufactured for use as research reagents, diagnostic agentsor potential and definite therapeutic agents. The term includesoligomers comprising of a single strand nucleic acid or a double strandnucleic acid.

The term “2′-modified” as used herein refers to a β-D-ribonucleoside orβ-D-ribonucleotide comprising of naturally occurring nucleobases havingthe 2′-OH group replaced by H, F, O—CH3 or other substituents known inthe art.

The term “2′-OH-nucleotide” as used herein refers to β-D-ribonucleotidecomprising of naturally occurring nucleobases having a 2′-OH group.

The term “5′-phosphate” as used herein refers to the formula—O—P(═O)(OH)OH. In another aspect the phosphate is modified that one ofthe O or OH groups is replaced by S and termed herein as“5′-phosphothioate”

The term “phosphorothioate” as used herein refers to a internucleotidelinkage in which one of the non-bridging oxygens is replaced by sulfur.

The term “delivery polymer” as used herein refers to polymers suitablefor functional delivery of a biologically active substance. In thecontext of the present invention the delivery polymer is eithercovalently attached to or coadministered with the biologically substanceconjugated to the compounds described herein and mediates endosomalescape after internalization into the cell and uptake into the endosome.The term “polymer” in this context means any compound that is made up oftwo or more monomeric units covalently bonded to each other, where themonomeric units may be the same or different, such that the polymer maybe a homopolymer or a heteropolymer. Representative polymers includepeptides, polysaccharides, nucleic acids and the like, where thepolymers may be naturally occurring or synthetic. Non-limiting examplesof delivery polymers are for example reviewed in INTERNATIONAL JOURNALOF PHARMACEUTICAL RESEARCH AND DEVELOPMENT,October—2010/Volume—2/Issue—8/Article No—2. Non-limiting examples ofdelivery polymers useful for delivery of nucleic acids are disclosed inEP applications 10165502.5 and 10191030.5, PCT publication WO2008/0022309 and U.S. provisional application 61/307,490 and referencescited herein; which are all included by reference.

As used herein, “pharmaceutical composition” includes the conjugates ofthe invention, a pharmaceutical carrier or diluent and any other mediaor agent necessary for formulation.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A conjugate of the present invention can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. To administer a conjugate of the invention by certainroutes of administration, it may be necessary to coat the conjugatewith, or co-administer the conjugate with, a material to prevent itsinactivation. For example, the conjugate may be administered to asubject in an appropriate carrier or a diluent. Pharmaceuticallyacceptable diluents include saline and aqueous buffer solutions.Pharmaceutical carriers include sterile aqueous solutions or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

These carriers may also contain adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents. Prevention of presenceof microorganisms may be ensured both by sterilization procedures,supra, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the conjugates ofthe present invention, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The pharmaceutical composition must be sterile and fluid to the extentthat the composition is deliverable by syringe. In addition to water,the carrier preferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 shows Co-Administration of siRNA-conjugates comprising thecompounds of formula (I) or (Ia) and a delivery polymer in vivo.

FIG. 2 shows Co-Administration of siRNA-conjugates comprising thecompounds of formula (I) or (Ia) and a delivery polymer in vivo.

FIG. 3 shows Co-Administration of siRNA-conjugates comprising thecompounds of formula (I) or (Ia) and a delivery polymer in vivo.

FIG. 4 shows Co-Administration of siRNA-conjugates comprising thecompounds of formula (I) or (Ia) and a delivery polymer in vivo.

FIG. 5a shows antisense strand mediated gene silencing with fully2′-modified siRNAs. COS7 cells were cotransfected with EGFP-directedsiRNAs at 3 nM and psiCHECK2-AT. The knockdown activity of the siRNAswas assessed by measuring renilla versus firefly luciferase activityfrom the reporter construct. siRNAs were sorted by knockdown activity ofunmodified (2-19-2) reference siRNAs.

FIG. 5b shows sense strand mediated gene silencing with fully2′-modified siRNAs. COS7 cells were cotransfected with EGFP-directedsiRNAs at 3 nM and psiCHECK2-ST. The knockdown activity of the siRNAswas assessed by measuring luciferase expression from the reporterconstruct. siRNAs were sorted by knockdown activity of unmodified(2-19-2) reference siRNAs.

FIG. 6a shows reduction of serum FVII activity in non-human primatesupon intravenous injection of various 2′-modified siRNAs covalentlyattached to a delivery polymer.

FIG. 6b shows the development of the prothrombin time in non-humanprimates upon treatment with 2′-modified siRNAs covalently conjugated toa delivery polymer.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

EXAMPLES Example 1 Step 1:3-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-propylamine

The title amine was prepared from its nitrile precursor according to aliterature protocol [Lollo et al, WO2001/070415].

Step 2:N-{3-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-propyl}-succinamicacid

In a 2 μL round-bottomed flask,3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propan-1-amine(21.15 g, 47.7 mmol, Eq: 1.00) and Huenig's base (12.3 g, 16.6 ml, 95.3mmol, Eq: 2.00) were combined with AcOEt (845 ml) to give a colorlesssolution. Dihydrofuran-2,5-dione (4.77 g, 47.7 mmol, Eq: 1.00) in THF(42 ml) was added and the reaction mixture was stirred at ambienttemperature over night=>white suspension. All volatiles were removed i.v., the residue dissolved in CH2Cl2, the organic layer washed with NH4Cland brine, dried over Na2SO4, and evaporated to dryness. The crudeproduct was dissolved in CH3CN/H2O and lyophilized to yield 29.8 g ofthe title compound as fluffy powder. MS (ISP): (M−H) 542.5.

Step 3:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-3-(4-nitrophenyl)-1-oxopropan-2-yl)succinamide

In a 10 mL round-bottomed flask, the above prepared4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanoicacid (106 mg, 184 μmol, Eq: 1.00),(S)-2-((S)-2-amino-3-(4-nitrophenyl)propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenylmethylamino)hexanamide(132 mg, 184 μmol, Eq: 1.00), HOAt (25.0 mg, 184 μmol, Eq: 1.00) and EDChydrochloride (35.3 mg, 184 μmol, Eq: 1.00) were mixed together inCH2Cl2 (1.8 ml) to give a yellow solution. Huenig's Base (47.5 mg, 64.2μl, 368 μmol, Eq: 2.00) was added and the reaction stirred at ambienttemperature over night. TLC indicated the consumption of startingmaterial. All volatiles were removed i. V. and the crude productpurified by flash chromatography SiO2/7% MeOH/0.1% NEt3 in CH2Cl2 toproduce 128 mg of the title compound as light yellow solid. MS: expectedmass: 1240.7552, found mass: 1240. 7518.

Step 4

In a 10 mL round-bottomed flask, the above preparedN1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-3-(4-nitrophenyl)-1-oxopropan-2-yl)succinamide(126 mg, 101 μmol, Eq: 1.00) and Huenig's base (39.3 mg, 53.2 μl, 304μmol, Eq: 3.00) were combined with CH2Cl2 (1.4 ml) and DMF (1.0 ml) togive a yellow suspension; bis(4-nitrophenyl) carbonate (46.3 mg, 152μmol, Eq: 1.50) was added and the reaction allowed to proceed overnight. The mixture was poured onto crashed ice, extracted 2× with AcOEt,washed with H2O, dried over Na2SO4, and evaporated to dryness. Aftertrituration with ˜10 ml of diethyl ether, 99 mg of the title product wasobtained as an off-white solid. MS: expected mass: 1405.7614, foundmass: 1405.7518.

The necessary dipeptide building block for step 3 was prepared asfollows:

Step a:(S)-2-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-(4-nitro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid

In a 25 mL round-bottomed flask,(S)-2-amino-6-((4-methoxyphenyl)diphenylmethyl-amino)hexanoic acid(Bioconjugate Chem. 2002, 13, 855-869, 968 mg, 2.31 mmol, Eq: 1.00) wasdissolved in CH2Cl2 (20 ml) to give a light yellow solution. Huenig'sbase (897 mg, 1.21 ml, 6.94 mmol, Eq: 3.00) and trimethylchlorosilane(528 mg, 621 μl, 4.86 mmol, Eq: 2.10) were added and the reactionmixture was stirred for 15 min.

In a second 50 mL round-bottomed flask,(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-nitrophenyl)propanoicacid (1 g, 2.31 mmol, Eq: 1.00) was dissolved in DMF (20 ml) to give acolorless solution. Huenig's base (359 mg, 485 μl, 2.78 mmol, Eq: 1.20)and TPTU [125700-71-2](687 mg, 2.31 mmol, Eq: 1.00) were added and thereaction mixture was stirred for 20′. The solution from the first flaskcontaining the corresponding silyl ester monosilylamine was added andthe reaction was stirred for another 3 hours. The mixture was pouredonto crashed ice/NH₄Cl, extracted 2× with AcOEt, washed with H2O andbrine, dried over Na2SO4, and evaporated to dryness. Flashchromatography SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 afforded 1.38 g of thetitle compound as brownish foam. MS (ISP): (M+H) 833.5, (M+Na) 855.4.

Step b:[(S)-1-((S)-1-(4-Hydroxymethyl-phenylcarbamoyl)-5-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-pentylcarbamoyl)-2-(4-nitro-phenyl)-ethyl]-carbamicacid 9H-fluoren-9-ylmethyl ester

In a 250 mL pear-shaped flask, the above synthesized(S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-nitrophenyl)propanamido)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanoicacid (1.38 g, 1.66 mmol, Eq: 1.00), (4-aminophenyl)methanol (204 mg,1.66 mmol, Eq: 1.00), HOAt (226 mg, 1.66 mmol, Eq: 1.00) and EDChydrochloride (318 mg, 1.66 mmol, Eq: 1.00) were dissolved in CH2Cl2(16.6 ml) to give a yellow solution. Huenig's base (428 mg, 579 μl, 3.31mmol, Eq: 2.00) was added and the reaction allowed to proceed overnight. The mixture was poured onto crashed ice/NH₄Cl (pH ˜7), extracted2× with AcOEt, washed with H2O, dried over Na2SO4, and evaporated todryness. The crude product was triturated with diethyl ether (1×50 mL);the resultant solid was filtered off and dryed to yield 1.214 g of thetitle compound as light-brown solid. MS (ISP): (M+H) 938.7.

Step c:(S)-2-[(S)-2-Amino-3-(4-nitro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide

In a 50 mL round-bottomed flask, the above prepared[(S)-1-((S)-1-(4-hydroxymethyl-phenylcarbamoyl)-5-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-pentylcarbamoyl)-2-(4-nitro-phenyl)-ethyl]-carbamicacid 9H-fluoren-9-ylmethyl ester (1.214 g, 1.29 mmol, Eq: 1.001) wascombined with THF (19 ml) to give a brown solution. At 0°, diethylamine(1.77 g, 2.49 ml, 24.2 mmol, Eq: 18.70) was added. The reaction wasstirred at ambient temperature for 3 h when MS indicated thedisappearance of the starting material. All volatiles were evaporated i.V.; ensuing flash chromatography SiO2/0.1% NEt3 in CH2Cl2=>10% MeOH/0.1%NEt3 in CH2Cl2, followed by a second flash chromatography SiO2/5%MeOH/0.1% NEt3 in CH2Cl2 afforded 502 mg of the title compound as lightbrown foam. MS: expected mass: 715.337, found mass: 715.3362.

Example 2O-Benzyl-N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-tyrosyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-amino-3-(4-benzyloxy-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-(benzyloxy)phenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1466.8182,found mass: 1466.8136.

Example 3N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-4-cyano-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-amino-3-(4-cyano-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-cyanophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1385.7716,found mass: 1385.7696.

Example 43,4-Dichloro-N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-amino-3-(3,4-dichloro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(3,4-dichlorophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1428.6984,found mass: 1428.695.

Example 54-Chloro-N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-((S)-2-amino-3-(4-chlorophenyl)propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideinstead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)-carbonylamino)-3-(4-chlorophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1394.7373,found mass: 1394.7342.

Example 64-{[(2S)-2-{[(2S)-2-[(4-{[3-({(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl}oxy)propyl]amino}-4-oxobutanoyl)amino]-3-(naphthalen-1-yl)propanoyl]amino}-6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}hexanoyl]amino}benzyl4-nitrophenyl carbonate (non-preferred name)

Was prepared in analogy to Example 1, but using in step 3(S)-2-((S)-2-amino-3-naphthalen-1-yl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(naphthalen-1-yl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1410.792,found mass: 1410.7918.

Example 7N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-4-fluoro-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-amino-3-(4-fluoro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)-hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-fluorophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1378.7669,found mass: 1378.7609.

Example 8N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-2-fluoro-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-Amino-3-(2-fluoro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)-hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(2-fluorophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1378.7669,found mass: 1378.7689.

Example 9N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-3-fluoro-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-[(S)-2-amino-3-(3-fluoro-phenyl)-propionylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide instead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)-hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(3-fluorophenyl)propanoicacid as described above in steps a]-c]. MS: expected mass: 1378.7669,found mass: 1378.7659.

Example 10 Step 1:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(4-fluorophenyl)-4-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-4-oxobutan-2-yl)succinamide

In a 10 mL round-bottomed flask, the above prepared4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanoicacid (109 mg, 188 μmol, Eq: 1.00),(S)-2-[(S)-3-amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide (132 mg, 188 μmol, Eq: 1.00), HOAt(25.6 mg, 188 μmol, Eq: 1.00) and EDC hydrochloride (36.1 mg, 188 μmol,Eq: 1.00) were mixed together in CH2Cl2 (2 ml) to give a yellowsolution. Huenig's Base (48.7 mg, 64.1 μl, 377 μmol, Eq: 2.00) was addedand the reaction stirred at ambient temperature over night. TLCindicated the consumption of starting material. All volatiles wereremoved i. V. and the crude product purified by flash chromatographySiO2/5% MeOH/0.1% NEt3 in CH2Cl2 to yield 197 mg of the title compoundas off-white solid. MS: expected mass: 1227.7763, found mass: 1227.7714.

Step 2:4-((S)-2-((S)-3-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)-4-(4-fluorophenyl)butanamido)-6-((4-methoxyphenyl)diphenylmethylamino)hexanamido)-benzyl4-nitrophenyl carbonate

In a 10 mL round-bottomed flask, the above preparedN1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(4-fluorophenyl)-4-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-4-oxobutan-2-yl)succinamide(196 mg, 160 μmol, Eq: 1.00) and Huenig's base (61.9 mg, 81.4 μl, 479μmol, Eq: 3.00) were combined with CH2Cl2 (1.6 ml) and DMF (0.8 ml) togive a yellow suspension; bis(4-nitrophenyl) carbonate (72.8 mg, 239μmol, Eq: 1.50) was added and the reaction allowed to proceed at ambienttemperature over night. The mixture was poured onto crashed ice/NH4Cl(pH ˜6), extracted 2× with AcOEt, washed with H2O and brine, dried overNa2SO4, and evaporated to dryness. After trituration with AcOEt/heptaneone obtained 123 mg of the title compound as light yellow solid. MS:expected mass: 1392.7825, found mass: 1392.7819.

The necessary dipeptidic building block for step 1 was prepared asfollows:

Step a:(S)-2-[(S)-3-(9H-Fluoren-9-ylmethoxycarbonylamino)-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid

In a 25 mL round-bottomed flask,(S)-2-amino-6-((4-methoxyphenyl)diphenylmethyl-amino)hexanoic acid(Bioconjugate Chem. 2002, 13, 855-869, 1040 mg, 2.48 mmol, Eq: 1.00) wasdissolved in CH2Cl2 (12.5 ml) to give a pale yellow solution. Huenig'sbase (961 mg, 1.27 ml, 7.44 mmol, Eq: 3.00) and trimethylchlorosilane(566 mg, 621 μl, 5.21 mmol, Eq: 2.10) were added and the reactionmixture was stirred at ambient temperature for 20 min.

In a second 50 mL round-bottomed flask,(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(4-fluorophenyl)butanoicacid (1040 mg, 2.48 mmol, Eq: 1.00) was dissolved in DMF (12.5 ml) togive a colorless solution. Huenig's base (385 mg, 506 μl, 2.98 mmol, Eq:1.20) and TPTU [125700-71-2] (737 mg, 2.48 mmol, Eq: 1.00) were addedand the reaction mixture was stirred for 15 min. The solution from thefirst flask containing the corresponding silyl ester monosilylamine wasadded and the reaction was stirred for another 3 hours at ambienttemperature. The mixture was poured onto crashed ice/NH₄Cl, extracted 2×with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporatedto dryness. Flash chromatography SiO2/5% MeOH/0.1% NEt3 in CH2Cl2afforded 2.10 g of the title compound as yellow foam. MS (ISP): (M+H)820.6.

Step b:{(S)-2-(4-Fluoro-phenyl)-1-[((S)-1-(4-hydroxymethyl-phenylcarbamoyl)-5-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-pentylcarbamoyl)-methyl]-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester

In a 250 mL pear-shaped flask, the above synthesized{(S)-2-(4-fluoro-phenyl)-1-[((S)-1-(4-hydroxymethyl-phenylcarbamoyl)-5-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-pentylcarbamoyl)-methyl]-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester (2.10 g, 2.56 mmol, Eq: 1.00),(4-aminophenyl)methanol (315 mg, 2.55 mmol, Eq: 1.00), HOAt (349 mg,2.56 mmol, Eq: 1.00) and EDC hydrochloride (491 mg, 2.56 mmol, Eq: 1.00)were dissolved in CH2Cl2 (12.5 ml). Huenig's base (662 mg, 871 μl, 5.21mmol, Eq: 2.00) was added and the reaction allowed to proceed overnight. The mixture was poured onto crashed ice/NH₄Cl (pH ˜7), extracted2× with AcOEt, washed with H2O and brine, dried over Na2SO4, andevaporated to dryness. The crude product was triturated with diethylether (1×50 ml); the resultant solid was filtered off and dryed to yield0.796 g of the title compound as light-brown solid. MS (ISP): (M+H)925.6.

Step c:(S)-2-[(S)-3-Amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide

In a 50 mL round-bottomed flask, the above prepared{(S)-2-(4-fluoro-phenyl)-1-[((S)-1-(4-hydroxymethyl-phenylcarbamoyl)-5-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-pentylcarbamoyl)-methyl]-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester (793 mg, 857 mol, Eq: 1.001) wascombined with THF (12 ml) to give a brownish solution. At 0°,diethylamine (1.13 g, 1.59 ml, 15.4 mmol, Eq: 18) was added. Thereaction was stirred at ambient temperature over night. The mixture waspoured onto crashed ice/NH₄Cl (pH ˜7), extracted 2× with AcOEt, washedwith H2O and brine, dried over Na2SO4, and evaporated to dryness. Flashchromatography SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 yielded 500 mg of thetitle compound as off-white solid. MS: expected mass: 702.3581, foundmass: 702.3578.

Example 114-((S)-2-((S)-3-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)-4-phenylbutanamido)-6-((4-methoxyphenyl)diphenylmethylamino)hexanamido)benzyl4-nitrophenyl carbonate

Was prepared in analogy to Example 10, but using in step 1(S)-2-((S)-3-amino-4-phenylbutanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideinstead of(S)-2-[(S)-3-amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide as coupling partner. The former wasprepared from(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-phenylbutanoic acid asdescribed above in steps a]-c]. MS: expected mass: 1374.792, found mass:1374.7877.

Example 124-({N˜2˜-[(3S)-4-(4-chlorophenyl)-3-{[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]amino}butanoyl]-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-L-lysyl}amino)benzyl4-nitrophenyl carbonate

Was prepared in analogy to example 10, but using in step 1(S)-2-((S)-3-amino-4-(4-chlorophenyl)butanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)-diphenylmethylamino)hexanamideinstead of(S)-2-[(S)-3-amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide as coupling partner. The former wasprepared from(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(4-chlorophenyl)-butanoicacid as described above in steps a]-c]. MS (ISP): (M+H) 1409.9.

Example 13N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-O-methyl-L-tyrosyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Was prepared in analogy to Example 1, but using in step 3(S)-2-((S)-2-amino-3-(4-methoxyphenyl)propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)-diphenylmethylamino)hexanamideinstead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. The former was prepared from(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-methoxyphenyl)propanoicacid as described above in steps a]-c] of example 1. MS (ISP): (M+H)1391.9.

Example 14N-[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-D-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-D-lysinamide

Was prepared in analogy to example 1, but using in step 3(R)-2-((R)-2-amino-3-phenyl-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenylmethyl)amino)-hexanamideinstead of(S)-2-((S)-2-amino-3-(4-nitrophenyl)-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideas coupling partner. This building block was synthesized from(R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6-aminohexanoic acid and(R)-2-amino-6-((4-methoxyphenyl)-diphenylmethylamino)hexanoic acid (seeBioconjugate Chem. 2002, 13, 885-869) as described above in steps a]-c].MS: expected mass: 1360.7763, found mass: 1360.7774.

Example 154-({N˜2˜-[(3S)-3-{[4-({3-[(3Beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]amino}-4-(4-cyanophenyl)butanoyl]-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-L-lysyl}amino)benzyl4-nitrophenyl carbonate

Was prepared in analogy to example 10, but using in step 1(S)-2-((S)-3-amino-4-(4-cyanophenyl)butanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanamideinstead of(S)-2-[(S)-3-amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide as coupling partner. The former wasprepared from(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(4-cyanophenyl)butanoicacid as described above in steps a]-c]. MS: expected mass: 1399.7872,found mass: 1399.7857.

Example 16N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

Step 1:(S)-2-((S)-2-Amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide

The building block(S)-2-((S)-2-amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide was prepared in analogy to theprocedure described in Bioconjugate Chem., Vol. 13, No. 4, 2002, 855-869MS (ISP): (M+H) 671.5

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)succinamide

TPTU [125700-71-2] (233 mg, 784 μmol, Eq: 1.00) was added to a solutionofN-{3-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-propyl}-succinamicacid (see example 1, step 2) (426 mg, 0.784 mmol, Eq: 1.00) and Huenig'sbase (304 mg, 411 μl, 2.35 mmol, Eq: 3) in DMF (10 ml). After 3 minutes(S)-2-((S)-2-amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoicacid (4-hydroxymethyl-phenyl)-amide (step 1) was added TLC at t=1 hshowed the reaction was complete. The solvent was removed under reducedpressure. The remaining residue was taken up in ethyl acetate andextracted with NaHCO₃ half sat. solution (1×), potassium hydrogenphthalate solution 0.05M (2×), water (1×) and brine (1×). The organicextract was dried over MgSO₄ and concentrated under reduced pressure.The crude material was purified by flash chromatography to obtain thetitled product (682 mg, 513, μmol) as a light brown solid. MS (ISP):(M+H) 1196.8

Step 3: Hünig's base (465 mg, 629 μl, 3.6 mmol, Eq: 6) was added to asolution of the previous alcohol (718 mg, 600 μmol, Eq: 1.00) andbis(4-nitrophenyl) carbonate (548 mg, 1.8 mmol, Eq: 3) in THF (20 ml).The yellow solution was stirred overnight at room temperature. Thesolvent was removed under reduced pressure. The remaining residue wastriturated with diethyl ether. The solid was collected by filtration,washed with ether and dried under reduced pressure to obtain the titlecompound (800 mg, 529 μmol) as a light brown solid. MS (ISP): (M+H)1361.9

Example 17 Step 1(S)-2-[(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propionylamino]-hexanoicacid

Commercially available L-Fmoc-Phe-OSu (0.969 g, 2.00 mmol, Eq: 1.00) wassuspended in a 1:1 v/v mixture of 1,2-dimethoxyethane and water (17 ml)and treated at 0° C. with L-norleucine (0.275 g, 2.10 mmoll, Eq: 1.05)and NaHCO₃ (0.185 g, 2.20 mmol, Eq: 1.10). The cooling bath was removedand the reaction allowed to proceed at ambient temperature for 14 h. Themixture was poured onto crashed ice/citric acid (pH ˜3), extracted 2×with ethyl acetate, washed with H2O and brine, dried over Na2SO4, andevaporated to dryness. Flash chromatography SiO2/AcOEt yielded 0.870 mgof the title compound as white solid. MS (ISP): (M+H) 501.2.

Step 2:{(S)-1-[(S)-1-(4-Hydroxymethyl-phenylcarbamoyl)-pentylcarbamoyl]-2-phenyl-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester

In a pear-shaped flask, the above synthesized(S)-2-[(S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propionylamino]-hexanoicacid (10.72 g, 21 mmol, Eq: 1.00), (4-aminophenyl)methanol (2.717 g, 22mmol, Eq: 1.03), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ) (7.994 g, 32 mmol, Eq: 1.50) were dissolved in CH2Cl2 (320 ml)and stirred overnight under a balloon of Ar. The mixture was poured ontocrashed ice/NH₄Cl, extracted 2× with AcOEt, washed with H2O, dried overNa2SO4, and the volume reduced to ˜300 ml. The precipitate was filteredoff and dryed to give 5.25 g of the title compound as light-brown solid.MS (ISP): (M+H) 606.3.

Step 3: (S)-2-((S)-2-Amino-3-phenyl-propionylamino)-hexanoic acid(4-hydroxymethyl-phenyl)-amide

In a round-bottomed flask, the above prepared{(S)-1-[(S)-1-(4-hydroxymethyl-phenylcarbamoyl)-pentylcarbamoyl]-2-phenyl-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester (4.738 g, 7.822 mmol, Eq: 1.0) wasdissolved in CH₂Cl₂ (28 ml). At 0°, diethylamine (28 ml, 19.80 g, 271mmol, Eq: 35) was added and the reaction mixture stirred at ambienttemperature overnight. All volatiles were evaporated i. V.; ensuingflash chromatography SiO2/CH2Cl2/10% MeOH, followed by crystallizationfrom AcOEt, yielded 2.116 g of the title compound as light browncrystals. MS (ISP): (M+H) 384.2.

Step 4:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxohexan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)succinamide

was prepared therewith in analogy to example 16 step 2. MS (ISP): (M+H)909.7 (M+Na) 931.8.

Step 5:N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-norleucinamide

was prepared therewith in analogy to example 16 step 3. MS expectedmass: 1073.6453, found mass 1073.642

Example 18N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-alanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

2-Chlorotrityl chloride resin (Novabiochem 01-64-0114, 100-200 mesh), 1%DVB (18 g, 21.6 mmol, Eq: 1.00) was swollen in DCM/DMF=1/1 (300 mL) forten minutes. The resin was drained and a solution of FMOC-4-aminobenzylalcohol (14.9 g, 43.2 mmol, Eq: 2) and pyridine (6.83 g, 6.99 ml, 86.4mmol, Eq: 4) in DCM/DMF=1/1 (300 mL) was added. The mixture was shakenovernight. The resin was drained and capped with a solution of 10%Hünig's Base in methanol (300 mL). The resin was washed with DMF and DCMand dried over night with HV to obtain 21.7 g resin. Determination ofthe load resulted in 0.41 mmoL/g.

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethylamino)-1-oxopropan-2-yl)succinamide

The resin from step 1 (1 g, 410 μmol, Eq: 1.00) was prewashed with DMF(2×) and treated with piperidine/DMF=1/4 (10 mL) for 5 and 10 minutes.The resin was washed alternately with DMF and IPA (3×10 mL). A solutionof Fmoc-Gly-OH (488 mg, 1.64 mmol, Eq: 4), TPTU (487 mg, 1.64 mmol, Eq:4) and Huenig's base (636 mg, 859 μl, 4.92 mmol, Eq: 12) in DMF (10 mL)was stirred for 5 minutes and then shaken with the resin for one hour.The resin was washed alternately with DMF and isopropyl alcohol (3×).

The following Fmoc cleavages and subsequent couplings of Fmoc-Ala-OH(511 mg, 1.64 mmol, Eq: 4) andN-{3-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-propyl}-succinamicacid (example 1, step 2) (892 mg, 1.64 mmol, Eq: 4) were performedaccordingly. The dried peptide resin was stirred for about 2×30 min inTFA 1%/DCM (2×20 mL). The reaction mixture was filtered and the resinwas washed with DCM. The filtrates were pooled and the solventsevaporated under vacuum. The crude material was triturated with diethylether (2×). After purification by flash chromatography, the product (84mg, 97.3 μmol) was obtained as a white solid. MS expected mass:776.5452, found mass 776.5455

Step 3: The above prepared alcoholN1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethylamino)-1-oxopropan-2-yl)succinamide[RO5545270](70 mg, 90.1 μmol, Eq: 1.00) and bis(4-nitrophenyl) carbonate(137 mg, 450 μmol, Eq: 5) under Argon at room temperature were dissolvedin DMF (4 ml) and treated with Huenig's base (34.9 mg, 47.2 μl, 270μmol, Eq: 3). and the mixture was allowed to react overnight. Thesolvent was removed in vacuo. The resulting solid was triturated withdiethylether. The solid was collected by filtration and washed withdiethyl ether. The product was dried in vacuo to obtain the titlecompound (84 mg, 80.2 μmol) as a light brown solid. MS expected mass:941.5514, found mass 941.5518

Example 19N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-methioninamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

was prepared in analogy to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-4-(methylthio)-1-oxobutan-2-ylamino)-4-methyl-1-oxopentan-2-yl)succinamide

was prepared in analogy to example 18, step 2, using Fmoc-Met-OH (609mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) asamino acids. The product (208 mg, 210 μmol) was obtained as a lightyellow solid. MS (ISP): (M+H) 893.6183

Step 3: was prepared in analogy to example 18, step 3. Afterpurification on silica gel, the title compound (161 mg, 137 μmol) wasobtained as light brown solid. MS expected mass: 1057.6174, found mass1057.6184

Example 20N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N˜1˜-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

was performed in analogy to example 18, step 1

Step 2:(S)-2-((S)-2-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)-4-methylpentanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Asn-OH (581mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) asamino acids. The product (87 mg, 89.4 μmol) was obtained as a lightyellow solid. MS expected mass: 875.6136, found mass 875.6133

Step 3: The titled compound was prepared in analogy to example 18, step3. After purification on silica gel (87 mg, 89.4 μmol) the titlecompound was obtained as light brown solid. MS expected mass: 1040.6198,found mass 1040.6188

Example 21N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-alanyl-N˜1˜-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

was performed in analogy to example 18, step 1

Step 2:(S)-2-((S)-2-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)propanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Asn-OH (581mg, 1.64 mmol, Eq: 4) and Fmoc-Ala-OH (511 mg, 1.64 mmol, Eq: 4) asamino acids. The product (140 mg, 159 μmol) was obtained as light yellowsolid. MS (ISP): (M+H) 834.8 (M+Na) 856.7.

Step 3: The title compound was prepared in analogy to example 18, step3. After purification on silica gel (169 mg, 152 μmol) it was obtainedas light brown solid. MS expected mass: 998.5729, found mass 998.5739

Example 22N˜2˜-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-asparaginyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed in analogy to example 18, step 1

Step 2:(S)-2-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)-N1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethyl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488mg, 1.64 mmol, Eq: 4) and Fmoc-Asn-OH (581 mg, 1.64 mmol, Eq: 4) asamino acids. The product (140 mg, 162 μmol) was obtained as white solid.MS expected mass: 819.551, found mass 819.5503

Step 3: The title compound was obtained in analogy to example 18, step 3(176 mg, 161 μmol) as light brown solid. MS expected mass: 984.5572,found mass 984.5489

Example 23N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed in analogy to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethylamino)-1-oxo-3-phenylpropan-2-yl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488mg, 1.64 mmol, Eq: 4) and Fmoc-Phe-OH (635 mg, 1.64 mmol, Eq: 4) asamino acids. The product (259 mg, 288 μmol) was obtained as white solid.MS expected mass: 852.5765, found mass 852.5754

Step 3: The title compound was obtained in analogy to example 18, step3. (280 mg, 247 μmol) as light brown solid. MS expected mass: 1017.5827,found mass 1017.5775

Example 24N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed in analogy to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethylamino)-4-methyl-1-oxopentan-2-yl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) asamino acids. The product (240 mg, 278 μmol) was obtained as a lightyellow solid. MS expected mass: 818.5921, found mass 818.5921

Step 3: The title compound was prepared in analogy to example 18, step3. After purification on silica gel, it (194 mg, 177 μmol) was obtainedas light yellow solid. MS expected mass: 983.5983 found mass 983.6004

Example 25N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-phenylalaninamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed in analogy to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxo-3-phenylpropan-2-ylamino)-4-methyl-1-oxopentan-2-yl)succinamide

Was prepared in analogy to example 18, step 2, using Fmoc-Phe-OH (635mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) asamino acids. The product (153 mg, 151 μmol) was obtained as light yellowsolid. MS expected mass: 908.6391 found mass 908.637.

Step 3: The title compound was prepared in analogy to example 18, step3. After purification on silica gel, it (117 mg, 98 μmol) was obtainedas white solid. MS expected mass: 1073.6453 found mass 1073.646

Example 26N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-phenylalaninamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed in analogy to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxo-3-phenylpropan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)succinamide

Was prepared in analogy to example 18, step 2, with Fmoc-Phe-OH (635 mg,1.64 mmol, Eq: 4) as amino acid. The product (240 mg, 204 μmol) wasobtained as light yellow solid. MS expected mass: 942.6234 found mass942.6218

Step 3: The title compound was prepared analogously to example 18, step3. After purification on silica gel, it (190 mg, 154 μmol) was obtainedas white solid. MS expected mass: 1107.6296 found mass 1107.6287

Example 27N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-leucinamide

Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-ChlorotritylResin

Was performed analogously to example 18, step 1

Step 2:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-4-methyl-1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)succinamide

Was prepared in analogy to example 18, step 2, with Fmoc-Leu-OH (1.59 g,4.5 mmol, Eq: 3) as amino acid. The product (254 mg, 284 μmol) wasobtained as white solid. MS expected mass: 874.6547 found mass 874.6527.

Step 3: The title compound was prepared in analogy to example 18, step3. After purification on silica gel it was obtained as white solid (178mg, 168 μmol). MS expected mass: 1039.6609 found mass 1039.6588

Example 28N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-alanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-alaninamide

Step 1:{(S)-1-[(S)-1-(4-Hydroxymethyl-phenylcarbamoyl)-ethylcarbamoyl]-ethyl}-carbamicacid 9H-fluoren-9-ylmethyl ester

A solution of Fmoc-Ala-Ala-OH (1 g, 2.61 mmol, Eq: 1.00) and(4-aminophenyl)methanol (483 mg, 3.92 mmol, Eq: 1.5) in THF (20 ml) wastreated with EEDQ (970 mg, 3.92 mmol, Eq: 1.5). The solution was stirredover night at room temperature. The mixture was diluted with 10%2-propanol/ethyl acetate (100 mL) and the solution was washed with KHSO45%/K2SO4 10% (2×), water (1×) and brine (1×), dried over MgSO4 andevaporated in vacuo. The residue was sonicated in diethyl ether forseveral minutes and the solid was collected by filtration to obtain theproduct (1.27 g, 1.2 mmol) as light brown solid. MS (ISP): (M+H) 488.3

Step 2:(S)-2-Amino-N—[(S)-1-(4-hydroxymethyl-phenylcarbamoyl)-ethyl]-propionamide

The compound was prepared in analogy to example 1 step c to obtain theproduct (245 mg, 877 μmol) as light yellow solid. MS (ISP): (M+H) 266.3,(M+Na) 288.2 (2M+H) 531.3

Step 3:N1-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propyl)-N4-((S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-yl)succinamide

The compound was prepared in analogy to example 16 step 2 (165 mg, 198μmol) as light brown solid. MS expected mass: 790.5608, found mass790.5587.

Step 4: The title compound was prepared in analogy to example 18, step3. After purification on silica gel it was obtained as white solid (99mg, 98.4 μmol). MS expected mass: 955.567, found mass 955.5651.

Example 29N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-isoleucyl-N˜1˜-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide

Step 1:(S)-2-[(2S,3S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methyl-pentanoylamino]-succinamicacid

2-Chlorotrityl chloride resin (5 g, 7.5 mmol, Eq: 1.00) was swollen inDCM and then treated with a solution of Fmoc-Asn(Trt)-OH (8.95 g, 15.0mmol, Eq: 2) and Huenig's base (3.88 g, 5.1 ml, 30.0 mmol, Eq: 4) in DCMovernight. The resin was washed with DCM and capped with a solution of10% Huenig's base in methanol. Coupling of Fmoc-Ile-OH (5.3 g, 15.0mmol, Eq: 2) with TPTU (4.46 g, 15.0 mmol, Eq: 2) and Huenig's base(3.88 g, 5.1 ml, 30.0 mmol, Eq: 4) according to standard solid phasepeptide synthesis. The product was cleaved from the resin with acocktail of TFA/Water/triisopropylsilane (95/2.5/2.5 v/v/v) for twohours at room temperature. The resin was filtered and the filtrate wasconcentrated under reduced pressure to a small volume. After triturationwith diethyl ether, the product was filtered and dried in vacuum toobtain the product (2.85 g, 5.79 mmol) as white solid. MS expected mass:467.2056, found mass 467.2056

Step 2:{(1S,2S)-1-[2-Carbamoyl-1-((S)-4-hydroxymethyl-phenylcarbamoyl)-ethylcarbamoyl]-2-methyl-butyl}-carbamicacid 9H-fluoren-9-ylmethyl ester

The compound was prepared in analogy to example 28 step 1 (620 mg, 336μmol) as light yellow solid.

Step 3:(S)-2-((2S,3S)-2-Amino-3-methyl-pentanoylamino)-N*1*-(4-hydroxymethyl-phenyl)-succinamide

The compound was prepared in analogy to example 1 step c (100 mg, 228μmol) as light yellow solid.

Step 4:(S)-2-((2S,3S)-2-(4-(3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)propylamino)-4-oxobutanamido)-3-methylpentanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide

The compound was prepared in analogy to example 16 step 2 (89 mg, 91.4μmol) as light yellow solid.

Step 5: The compound from the previous step was reacted to the titlecompound analogously to example 18, step 3. After purification on silicagel, it (42 mg, 36.3 μmol) was obtained as a light brown solid. MSexpected mass: 1040.6198, found mass 1040.6177

Example 30N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-D-lysinamide

The compound was prepared in analogy to example 16 step 1, starting withFmoc-D-Lys(Boc)-OH, (158 mg, 116 μmol) as light brown solid. MS (ISP):(M+H) 1362.8 (M+Na) 1383.8

Example 31N-{15-[(3beta)-cholest-5-en-3-yloxy]-4,15-dioxo-8,11-dioxa-5,14-diazapentadecan-1-oyl}-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

The title compound was prepared analogous to example 16 using acholesterol-oligo-PEG derivative in step 2 of the synthesis. MS (ISP):(M+H) 1479.8

The necessary cholesterol-PEG intermediateN-[2-(2-{2-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-ethoxy}-ethoxy)-ethyl]-succinamicacid for step 2 was prepared as follows:

Step a: {2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester

A solution of cholesteryl chloroformate (1 g, 2.23 mmol) in 25 mLdichloromethane was added dropwise under stirring to a solution of2,2′-(ethylenedioxy)bis-(ethylamine) (495 mg, 3.34 mmol) in 75 mLdichloromethane. The reaction was stirred overnight at room temperature.The reaction was diluted with dichloromethane and extracted with water.The organic extract was dried over anhydrous MgSO4 dihydrate, filteredand evaporated. After purification on amino-modified silica gel (eluent:MeCl2→MeCl2/MeOH=975:25 v/v) the product (615 mg) was obtained as awhite, waxy solid. MS (ISP): (M+H) 561.5

Step b:N-[2-(2-{2-[(3S,8S,9S,10R,13R,14S,17R)-17-((R)-1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-ethoxy}-ethoxy)-ethyl]-succinamicacid

The amine from step a (480 mg, 0.856 mmol) and triethylamine (0.13 mL,0.94 mmol) were dissolved in 5 mL dichloromethane. After adding succinicanhydride (90 mg, 0.9 mmol) the solution was stirred overnight at roomtemperature. TLC check showed still some starting material. Moresuccinic anhydride (20 mg, 0.2 mmol) was added. After stirring thereaction for another 3 hours at room temperature, it was diluted withdichloromethane and washed with a 5% KHSO4/10% K2SO4 mixture. Theorganic extract was dried over anhydrous MgSO4-dihydrate, filtered andevaporated in vacuo to obtain the desired acid (490 mg, 0.667 mmol). MS(ISP): (M+H) 661.5

Example 32N-{30-[(3beta)-cholest-5-en-3-yloxy]-4,30-dioxo-8,11,14,17,20,23,26-heptaoxa-5,29-diazatriacontan-1-oyl}-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

The title compound was prepared analogous to example 16 using acholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M+H)1699.9

The necessary cholesterol-PEG intermediate1-[(3beta)-cholest-5-en-3-yloxy]-1,27-dioxo-5,8,11,14,17,20,23-heptaoxa-2,26-diazatriacontan-30-oicacid for step 2 was prepared as follows:

Step a: tert-butyl[25-({(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl}oxy)-25-oxo-3,6,9,12,15,18,21-heptaoxa-24-azapentacos-1-yl]carbamate

Cholesteryl chloroformate (476 mg, 1.06 mmol) and triethylamine (155 uL,1.113 mmol) were dissolved in 5 mL dichloromethane. Then a solution ofalpha-amino-omega-boc-amino-octa(ethylene glycol) (497 mg, 1.06 mmol)dissolved in 1 mL dichloromethane was added. The solution was stirredover night at room temperature and diluted with dichloromethane andextracted with a KHSO4 5%/K2SO4 10% aqueous mixture. The organic extractwas dried over anhydrous MgSO4, filtered and evaporated in vacuo. Afterpurification on silica gel (eluent: MeCl2/MeOH=975:25→95:5 v/v) theproduct (530 mg, 0.571 mmol) was obtained as a colorless oil. MS (ISP):(M+NH4) 898.7

Step b:1-[(3beta)-cholest-5-en-3-yloxy]-1,27-dioxo-5,8,11,14,17,20,23-heptaoxa-2,26-diazatriacontan-30-oicacid

The previous Boc derivative (450 mg, 0.511 mmol) was dissolved in HCl 4Min dioxane (10.2 mL, 40.9 mmol). The solution was stirred at roomtemperature for 40 min. The solvent was removed in vacuo and theremaining white solid was dissolved in 5 mL dichloromethane and treatedwith triethylamine (32 uL, 0.229 mmol) and succinic anhydride (11.5 mg,0.114 mmol) overnight. More succinic anhydride (11 mg, 0.11 mmol, 0.2equiv.) was added and after 60 min the reaction was diluted withdichloromethane and washed with KHSO4 5%/K2SO4 10% buffer. The organicextract was dried over MgSO4 anhydrous, filtered and evaporated toobtain 390 mg of the desired product. MS (ISP): (M+H) 881.7

Example 33N-{66-[(3beta)-cholest-5-en-3-yloxy]-4,66-dioxo-8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62-nonadecaoxa-5,65-diazahexahexacontan-1-oyl}-L-phenylalanyl-N˜6˜-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide

The title compound was prepared analogous to example 16 using acholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M+H)2228.1

The necessary cholesterol-PEG intermediate1-[(3beta)-cholest-5-en-3-yloxy]-1,63-dioxo-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59-nonadecaoxa-2,62-diazahexahexacontan-66-oicacid for step 2 was prepared as follows:

Step a:(3beta)-cholest-5-en-3-yl(59-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57nonadecaoxanonapentacont-1-yl)carbamate

Alpha, omega-bis-amino 20 (ethylene glycol) (538 mg, 0.6 mmol) andtriethylamine (92 uL, 0.66 mmol) were dissolved in 15 mL drydichloromethane. A solution of cholesteryl chloroformate (270 mg, 0.6mmol) in 2 mL dry dichloromethane was added dropwise at roomtemperature. The solution was stirred overnight, then concentrated invacuo to a small volume and purified directly on silica gel (eluent:MeCl2/MeOH=95:5→9:4→4:1 v/v) to obtain the product (350 mg, 0.254 mmol)as a waxy solid. MS (ISP): (M+H) 1309.9

Step b:1-[(3beta)-cholest-5-en-3-yloxy]-1,63-dioxo-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59-nonadecaoxa-2,62-diazahexahexacontan-66-oicacid

The amine from step a (329 mg, 0.251 mmol), succinic anhydride (26.4 mg,0.264 mmol) and triethylamine (40 uL, 0.286 mmol) were dissolved in 5 mLdry dichloromethane. After adding more triethylamine (40 uL, 0.286mmol), the solution (pH>8) was stirred overnight at room temperature.The reaction was diluted with dichloromethane and washed twice with aKHSO₄ 5%/K₂SO₄ 10% aqueous mixture. The organic extract was dried overanhydrous MgSO₄, filtered and evaporated to obtain the product (260 mg,0.175 mmol) as a colorless, waxy solid. MS (ISP): (M+NH4) 1408.9

Example 34 General Procedure for the Preparation of RNA ConjugatesMaterials

Dimethyl sulfoxide (DMSO), N,N-Diisopropylethylamine (DIPEA) and sodiumacetate solution (3 M, pH 5.2) were purchased from Sigma Aldrich ChemieGmbH (Traufkirchen, Germany).

Triethylammonium acetate (TEAA) (2.0 M, pH 7.0) and Acetonitrile (ACN,HPLC quality) for RP-HPLC were purchased from Biosolve (Valkenswaard,Netherlands).

Ethanol (EtOH, p.a.) was purchased from Merck (Darmstadt, Germany).Purified water from a Optilab HF (Membra Pure, Germany) system was used.

Resource RPC 3 mL column (10×0.64 cm; 15 μm particle size) was purchasedfrom GE Healthcare (Freiburg, Germany).

HPLC purification was accomplished using an ÄKTA Explorer 100 system (GEHealthcare).

Synthesis of Amino-Modified RNA

RNA equipped with a hexylaminolinker at the 5′-end of the sense strandwas produced by standard phosphoramidite chemistry on solid phase at ascale of 1215 μmol using an ÄKTA Oligopilot 100 (GE Healthcare,Freiburg, Germany) and controlled pore glass as solid support (PrimeSynthesis, Aston, Pa., USA). RNA containing 2′-O-methyl nucleotides weregenerated employing the corresponding phosphoramidites, 2′-O-methylphosphoramidites and TFA-hexylaminolinker amidite (Sigma-Aldrich, SAFC,Hamburg, Germany). Cleavage and deprotection as well as purification wasachieved by methods known in the field (Wincott F., et al, NAR 1995, 23,14, 2677-84).

The amino-modified RNA was characterized by anion exchange HPLC (purity:96.1%) and identity was confirmed by ESI-MS ([M+H]1+ calculated: 6937.4;[M+H] 1+measured: 6939.0.

Sequence: 5′-(NH2C6)GGAAUCuuAuAuuuGAUCcAsA-3′ (SEQ ID NO: 283); u, c:2′-O-methyl nucleotides of corresponding RNA nucleotides, s:phosphorthioate.

General Experimental Conjugation Procedure

The title compounds of examples 1-33 were coupled via the amino-modifiedRNA according the following procedure:

RNA equipped with a C-6 aminolinker at the 5′-end (16.5 mg, 1equivalent) is dissolved in 500 μL DMSO and 150 μL water. Thep-Nitrophenylcarbonate derivative (10 equivalents) dissolved in 1 mLDMSO is added followed by 8 μL DIPEA. The reaction mixture is shaken at35° C. in the dark and monitored using RP-HPLC (Resource RPC 3 mL,buffer: A: 0.1M TEAA in water, B: 0.1M TEAA in 95% ACN, gradient: 3% Bto 100% B in 20 CV). Once the reaction is gone to completion the RNAconjugate is precipitated using sodium acetate (3 M) in EtOH at −20° C.For examples lacking a MMT protecting group in the dipeptide motif thecorresponding conjugates are purified using the conditions describedabove. Pure fractions are pooled and the material is precipitated usingsodium acetate/EtOH to give the desired RNA conjugate.

RNA conjugates containing a MMT protecting group in the dipeptidesequence are further processed according to the procedure given below.

General Procedure for MMT Cleavage

The crude RNA conjugate pellet is dissolved in 500 μL water and 1.5 mLsodium acetate buffer (3 M, pH 5.2 or 0.1M, pH 4.0). The solution isshaken for 2 days at 30° C. The reaction mixture is monitored usingRP-HPLC (Resource RPC 3 mL, buffer: A: 0.1M TEAA in water, B: 0.1M TEAAin 95% ACN, gradient: 3% B to 100% B in 20 CV). After complete cleavageof the MMT protecting group the RNA conjugate is directly purified usingthe conditions just mentioned above. Pure fractions are pooled and thedesired conjugate is precipitated using sodium acetate/EtOH.

As a control a RNA conjugate lacking the dipeptide motif wassynthesized. For this purpose cholesterol was attached to the 5′-end viaa linker described in the literature (Nature Biotech, 2007, 25, 1149).This conjugate is referred to as “non-cleavable”.

All the RNA conjugates were analyzed by RP HPLC for purity and identitywas confirmed by ESI MS (negative mode). Briefly, RP-HPLC was performedon a Dionex Ultimate system (Dionex, Idstein, Germany) equipped with aXBridge C₁₈ column (2.5×50 mm, 2.5 μm particle size, Waters, Eschborn,Germany) at 65° C. column temperature. Gradient elution was performedusing 100 mM hexafluoroisopropanol (HFIP) and 16 mM triethylamine in 1%methanol as eluent A and in 95% methanol as eluent B (1% B to 18% B in30 minutes). UV detection was recorded at 260 nm. For mass spectrometricanalysis a ThermoFinnigan LCQ DecaXP ESI-MS system with micro-spraysource and ion trap detector was coupled online to the HPLC system.

Examples of specific compounds of formula (IIa) are disclosed intable 1. The resulting compounds are referred to “di-peptide containingcholesterol siRNA conjugates”, wherein the specific di-peptidecontaining cholesterol siRNA conjugates are further referred to as“Title compound example X—(NHC6)-(siRNA sequence)” and “siRNA with Titlecompound of Example X”.

siRNA Preparation

Antisense sequence: (SEQ ID NO: 154) 5′-uuGGAUcAAAuAuAAGAuUCcscsU-3′

u, c: 2′-O-methyl nucleotides of corresponding RNA nucleotides, s:phosphorthioate

The di-peptide containing cholesterol siRNA conjugates directed againstthe apolipoprotein B mRNA were generated by mixing an equimolar solutionof complementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 80-85° C. for 3minutes and cooled to room temperature over a period of 3-4 hours.Duplex formation was confirmed by native gel electrophoresis.

All prepared di-peptide containing cholesterol siRNA conjugates arelisted in table 2.

TABLE 1Di-peptide containing cholesterol siRNA conjugates (5′-3′) and analytical data.Key: lower case letters a, c, g, u, are 2′-O-Methyl nucleotides; A phosphorothioatelinkages is symbolized with a lower case “s”. (NHC6) is the aminohexyl linkerincorporatd at the 5′-end of the sense strand. Title compound Base fromsequence Mol Mol Purity example SEQ ID mass mass (%) No. NO: calc. exp.(RP) 16 (Title compound Ex 16)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7885.77887.5 94.4 31 (Title compound Ex 16)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2848003.8 8003.3 97.3 33 (Title compound Ex 33)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 8752.7 8752.4 97.6 32(Title compound Ex 32)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 8223.1 8226.597.3 17 (Title compound Ex 17)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7870.77873.5 90.6 30 (Title compound Ex 30)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847884.8 7888.8 95.2 27 (Title compound Ex 27)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7840.0 7840.0 94.8 28(Title compound Ex 28)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7755.4 7754.993.2 29 (Title compound Ex 29)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7840.47839.9 87.2 1 (Title compound Ex 1) (NHC6)GGAAUCuuAuAuuuGAUCcASA 2847931.1 7935.2 98.2 2 (Title compound Ex 2)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7992.2 7995.0 96.7 3(Title compound Ex 3)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7911.1 7913.8 98.24 (Title compound Ex 4)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7955.1 7958.598.0 5 (Title compound Ex 5)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7920.57923.9 97.2 6 (Title compound Ex 6)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847936.1 7939.6 98.5 7 (Title compound Ex 7)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7904.2 7905.5 95.1 8(Title compound Ex 8)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7904.2 7908.7 98.89 (Title compound Ex 9)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7904.2 7906.798.7 10 (Title compound Ex 10)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7918.27921.0 95.4 11 (Title compound Ex 11)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847902.0 7901.5 98.7 12 (Title compound Ex 12)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7934.54 7936.5 94.4 13(Title compound Ex 13)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7916.09 7917.996.5 14 (Title compound Ex 14)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7886.077888.3 94.9 24 (Title compound Ex 24)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847781.8 7783.4 97.2 23 (Title compound Ex 23)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7815.8 7817.3 95.2 22(Title compound Ex 22)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7781.8 7783.990.5 26 (Title compound Ex 26)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7905.97907.0 96.4 25 (Title compound Ex 25)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847871.9 7873.2 96.1 20 (Title compound Ex 20)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7840.7 7840.0 95.9 19(Title compound Ex 19)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7857.8 7856.697.3 18 (Title compound Ex 18)(NHC6)GGAAUCuuAuAuuuGAUCcASA 284 7741.67741.1 93.9 21 (Title compound Ex 21)(NHC6)GGAAUCuuAuAuuuGAUCcASA 2847798.6 7797.8 87.6 15 (Title compound Ex 15)(NHC6)GGAAUCuuAuAuuuGAUCcASA284 7927.1 7926.8 97.2

TABLE 2Di-peptide containing cholesterol siRNA conjugates. The last entry(SEQ ID NO pair 266/154) represents a siRNA conjugate lacking thedi-peptide motif. Key: lower case letters a, c, g, u, are 2′-O-Methylnucleotides; A phosphorothioate linkages is symbolized with a lowercase “s”. (NHC6) is the aminohexyl linker incorporated at the5′-end of the sense strand. SEQ ID SEQ ID No Sense sequence (5′-3′) NoAntisense sequence (5′-3′) 284 (Title compound Ex 16)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 31)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 33)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 32)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 17)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 30)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 27)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 28)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 29)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 1)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 2)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 3)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 4)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 5)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 6)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 7)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 8)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 9)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 10)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 11)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 12)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 13)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 14)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 24)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 23)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 22)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 26)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 25)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 20)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 19)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 18)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 284(Title compound Ex 21)(NHC6) 154 uuGGAUcAAAuAuAAGAuUCcscsUGGAAUCuuAuAuuuGAUCcAsA 284 (Title compound Ex 15)(NHC6) 154uuGGAUcAAAuAuAAGAuUCcscsU GGAAUCuuAuAuuuGAUCcAsA 285(Chol)GGAAUCuuAuAuuuGAUCcAcA 154 uuGGAUcAAAuAuAAGAuUCcscsU

Example 35 In Vivo Experiments

Co-Administration of Di-Peptide Containing Cholesterol siRNA Conjugatesand Delivery Polymer In Vivo

Six to eight week old mice (strain C57BL/6 or ICR, ˜18-20 g each) wereobtained from Harlan Sprague Dawley (Indianapolis Ind.). Mice werehoused at least 2 days prior to injection. Feeding was performed adlibitum with Harlan Teklad Rodent Diet (Harlan, Madison Wis.).

Mice (n=3 per group) were injected with a mixture of 0.2 mL solution ofdelivery polymer and 0.2 ml di-peptide containing cholesterol siRNAconjugates. The injected dose was, unless otherwise stated, 15 mg/kg forthe delivery polymer and 0.1 mg/kg with respect to the di-peptidecontaining cholesterol siRNA conjugates. Solutions were injected byinfusion into the tail vein. 48 hours post injection serum ApoB levelswere measured relative to isotonic glucose treated animals according tothe procedure below.

Serum ApoB Levels Determination.

Mice were fasted for 4 h before serum collection by submandibularbleeding. Serum ApoB protein levels were determined by standard sandwichELISA methods. Briefly, a polyclonal goat anti-mouse ApoB antibody and arabbit anti-mouse ApoB antibody (Biodesign International) were used ascapture and detection antibodies respectively. An HRP-conjugated goatanti-rabbit IgG antibody (Sigma) was applied afterwards to bind theApoB/antibody complex. Absorbance of tetramethyl-benzidine (TMB, Sigma)colorimetric development was then measured by a Tecan Safire2 (Austria,Europe) microplate reader at 450 nm.

In FIG. 1 various di-peptide containing cholesterol siRNA conjugateswere benchmarked against the same siRNA conjugated to cholesterol butlacking the cleavable motif elaborated earlier in this section. Theeffect of this siRNA conjugate (SEQ ID NO pair 266/154, “non-cleavablecontrol”) on serum ApoB levels was set to 1 in order to evaluate theinfluence of the di-peptide containing conjugates relative to thenon-cleavable control. Substituting the initially used Phe-Lys motif(siRNA with Title compound of Example 16) with the corresponding D-aminoacids (siRNA with Title compound of Example 14) or just replacing theLys with the unnatural enantiomer (siRNA with Title compound of Example30) yielded ApoB reduction less pronounced or equivalent to thenon-cleavable control siRNA. Replacing Lys by Gly (siRNA with Titlecompound of Example 23) or Phe by p-Methoxyphenylalanine (siRNA withTitle compound of Example 13) reduced the potency compared to siRNA withTitle compound of Example 16. Other di-peptide motifs containing siRNAconjugates were shown to be as efficacious as the original Phe-Lyscontaining conjugate.

FIG. 2 summarizes di-peptide containing cholesterol siRNA conjugatesthat were as efficacious or had improved efficacy compared to siRNA withTitle compound of Example 16 consisting of the Phe-Lys motif. All theseconjugates were significantly more active compared to the“non-cleavable” cholesterol siRNA conjugate SEQ ID NO pair 266/154. Thebest performing di-peptide containing cholesterol siRNA conjugates had afluorine modified phenyl ring in the Phy-Lys motif (siRNA with Titlecompound of Example 8, siRNA with Title compound Example 9) or had thephenylalanine substituted with beta-phenylalanine (siRNA with Titlecompound of Example 11) or a derivative thereof (siRNA with Titlecompound of Example 10).

Since di-peptide containing cholesterol siRNA conjugates with di-peptidemotifs consisting of D-amino acids are performing equal to thenon-cleavable control conjugate it is conceivable that the otherdi-peptide sequences are indeed cleaved by a protease activity in vivo.However, given the broad acceptance of different amino acids andderivatives thereof it is likely that more than one enzyme isparticipating in the cleavage reaction as suggested in the literature(Bioconjugate Chem. 2002, 13, 855).

As shown in FIG. 3, the incorporation of a Cathepsin cleavabledi-peptide motif (in this case Phe-Lys, siRNA with Title compound ofExample 16) between the siRNA and the small molecule ligand cholesterolboosts the potency of the siRNA conjugate compared to the straightcholesterol siRNA conjugate (SEQ ID NO pair 266/154). Further spacing ofthe cholesterol ligand from the di-peptide motif by means of PEG basedlinkers diminishes the potency proportional to the length of the PEGlinker.

In FIG. 4 the polymer dose was kept constant at 15 mg/kg. The siRNA dosewas titrated and the effect on serum ApoB content was measured. TheDi-peptide containing cholesterol siRNA conjugates containing thePhe-Lys (F-K) motif was significantly more potent compared to thecontrol conjugate lacking the di-peptide sequence.

Example 36 2′-Modified Oligoribonucleotide Synthesis

Oligoribonucleotides were synthesized according to the phosphoramiditetechnology on solid phase. Depending on the scale either an ABI 394synthesizer (Applied Biosystems) or an AKTA oligopilot 100 (GEHealthcare, Freiburg, Germany) was used. Syntheses were performed on asolid support made of controlled pore glass (CPG, 520 {acute over (Å)},with a loading of 75 μmol/g, obtained from Prime Synthesis, Aston, Pa.,USA). All 2′-modified RNA phosphoramidites as well as ancillary reagentswere purchased from SAFC (Hamburg, Germany). Specifically, the following2′-O-Methyl phosphoramidites were used:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-dimethoxytrityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,and5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite.The 2′-Deoxy-2′-fluoro-phosphoramidites carried the same protectinggroups as the 2′-O-methyl RNA amidites. All amidites were dissolved inanhydrous acetonitrile (100 mM) and molecular sieves (3 {acute over(Å)}) were added. To generate the 5′-phosphate the2-[2-(4,4′-Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramiditefrom Glen Research (Sterling, Va., USA) was used. In order to introducethe C-6 aminolinker at the 5′-end of the oligomers the6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramiditefrom Thermo Fisher Scientific (Milwaukee, Wis., USA) was employed. The5′-modifications were introduced without any modification of thesynthesis cycle. 5-Ethyl thiotetrazole (ETT, 500 mM in acetonitrile) wasused as activator solution. Coupling times were 6 minutes. In order tointroduce phosphorothioate linkages a 50 mM solution of3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT,obtained from AM Chemicals, Oceanside, Calif., USA) in anhydrousAcetonitrile/pyridine (1:1 v/v) was employed.

Example 37 Cleavage and Deprotection of Support Bound Oligomer

After finalization of the solid phase synthesis, the dried solid supportwas transferred to a 15 mL tube and treated with concentrated aqueousammonia (Aldrich) for 18 hours at 40° C. After centrifugation thesupernatant was transferred to a new tube and the CPG was washed withaqueous ammonia. The combined solutions were evaporated and the solidresidue was reconstituted in buffer A (see below).

Example 38 Purification of Oligoribonucleotides

Crude oligomers were purified by anionic exchange HPLC using a columnpacked with Source Q15 (GE Helthcare) and an AKTA Explorer system (GEHelthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mMEDTA, pH 7.4 (Fluka, Buchs, Switzerland) and contained 20% Acetonitrileand buffer B was the same as buffer A with the exception of 500 mMsodium perchlorate. A gradient of 22% B to 42% B within 32 columnvolumes (CV) was employed. UV traces at 280 nm were recorded Appropriatefractions were pooled and precipitated with 3M NaOAc, pH=5.2 and 70%Ethanol. Finally, the pellet was washed with 70% Ethanol.

Example 39 Annealing of Oligoribonucleotides to Generate siRNA

Complementary strands were mixed by combining equimolar RNA solutions.The mixture was lyophilized and reconstituted with an appropriate volumeof annealing buffer (100 mM NaCl, 20 mM sodium phosphate, pH 6.8) toachieve the desired concentration. This solution was placed into a waterbath at 95° C. which was cooled to rt within 3 h.

Example 40 In Vitro Activity of siRNAs Devoid of 2′-OH Residues

In order to investigate if siRNAs lacking any 2′-OH residues show potentin vitro knock down activity, we tested a panel of EGFP mRNA-targetedsiRNAs with different 2′-modification chemistries (SEQ ID pairs 31/32 to149/150, and see Table 3 for examples). The siRNAs were screened forsense and antisense activity with the Dual-Glo® Luciferase Assay System(Promega) using the psiCHECK2 vector (Promega) in COS7 cells (DSMZ,Braunschweig, Germany, cat. No. ACC-60). To address the silencingactivity conferred by sense and antisense strand we cloned eachcorresponding 19mer target site sequence as separate psiCHECK2 construct(psiCHECK2-AT for antisense activity, psiCHECK2-ST for sense activity)into the multiple cloning region located 3′ to the translational stopcodon of the synthetic Renilla luciferase. By using Lipofectamine 2000(Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) COS7 cellswere co-transfected with vector construct and 3 nM of the correspondingsiRNA complementary to the cloned target site. Successful siRNA-mediatedsilencing was determined 24 hours after transfection via the activity ofthe renilla luciferase normalized to firefly luciferase levels to taketransfection efficiency into account (see FIG. 5a for antisense activityand FIG. 5b for sense activity).

Table 3: Exemplary siRNA sequences and chemical modifications used fordetermination of in vitro knock down activity dependent on2′-modifications. Reference duplices and selected examples ofcorresponding modification variants used in this study. Xf indicates a2′-fluoro modification of the nucleotide X, small letters indicate a2′-O-methyl modification, underlined letters indicate a DNA nucleotide,all other capital letters indicate ribonucleotides. The letter “p”indicates a 5′-phosphate.

RNA duplices unmod 5′- UGCCCAUCCUGGUCGAGCUTT -3′ (SEQ ID NO: 286)3′- TTACGGGUAGGACCAGCUCGAp -5′ (SEQ ID NO: 287) F/OMe5′- UfgCfcCfaUfcCfuGfgUfcGfaGfcUfTsT -3′ (SEQ ID NO: 288)3′- TsTaCfgGfgUfaGfgAfcCfaGfcUfcGfap -5′ (SEQ ID NO: 289) F/DNA5′- UfGCfCCfAUfCCfUGfGUfCGfAGfCUfTsT -3′ (SEQ ID NO: 290)3′- TsTACfGGfGUfAGfGAfCCfAGfCUfCGfAp -5′ (SEQ ID NO: 291) DNA/OMe5′- UgCcCaUcCuGgUcGaGcUTsT -3′ (SEQ ID NO: 292)3′- TsTaCgGgUaGgAcCaGcUcGap -5′ (SEQ ID NO: 293)

It was found that the 5 most potent modified siRNAs (≧60% knock-down)were designed in an alternating 2′-fluoro/2′-O-methyls (2′F/2′-OMe)pattern. While conferring antisense activity, this chemistry fullyeliminated the activity of the corresponding sense strands, as shown bylack or minimal renilla luciferase activity for all tested 2′F/2′-OMevariants.

We concluded that such 2′F/2′-OMe pattern is promoting the siRNA'sintended antisense strand activity while undesired off-target effectscoming from the sense strand are fully suppressed. This design isspecifically preferable for siRNAs, that come with the need forprotection against 2′O-directed nucleolytic cleavage.

Example 41 Detection of DNAse II-Sensitive Sites by In Vitro Assay

An ion pairing (IP) reversed phase (RP) high performance liquidchromatography (HPLC) coupled to an electrospray ionization (ESI) massspectrometry (MS) or an anion exchange (AEX)-HPLC based method wasestablished to test the in vitro stability of selected single and doublestranded RNAs.

Method description: For stability analysis a 10 μM solution of eithersingle stranded or double stranded RNA was incubated at 37° C. in 5 mMsodium acetate buffer solution (pH 4.5) containing 0.8 or 8 units DNaseII (from bovine spleen, Type V, Sigma Aldrich). The incubation reactionwas stopped by adding a 100 mM triethyl ammonium acetate (TEAA)solution, shifting the pH to 7 and inactivating the DNase II enzyme.Analysis was done by either LC/MS combined with UV-detection or byAEX-HPLC with UV-detection. UV-detection traces at 260 nm were used forquantitative analysis, MS data served for cleavage site identificationwithin the RNA sequence.

-   A. IP-RP-HPLC was done employing a Waters XBridge C₁₈ column (2.5×50    mm, 2.5 μm particle size) at 65° C. column temperature. Gradient    elution was performed using 100 mM hexafluoroisopropanol (HFIP) and    16 mM triethylamine in 1% methanol as eluent A and composition A in    95% methanol as eluent B. A gradient from 1% B to 18% B in 30    minutes was employed.-   B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4×250 mm)    at 50° C. using a 20 mM phosphate buffer containing 10% ACN at    pH=11. Eluent B contained 1 M NaBr in eluent A. A gradient from 25    to 62% B in 18 minutes was employed.

TABLE 4 Duplexes and the remaining intact strands evaluated for theirstability against DNase II. Key: lower case letters a, c, g,u, are 2′-O-Methyl nucleotides; Upper case letters A, C, G,U followed by “f” indicates a 2′-fluoro nucleotide. Lower case “p”indicates a 5′-phosphate. (invdT) represents aninverted deoxythimidine (3′-3′-linked). A phosphorothioatelinkages is symbolized with a lower case “s”. dT is deoxy-thimidine. (NHC6) is the aminohexyl linker incorporated atthe 5′-end of the sense strand. % Intact % Intact SEQ strand SEQ strandID Sense strand after 6 ID Antisense strand after 6 NO sequence (5′-3′)hours NO sequence (5′-3′) hours 157 GGAuGAAGuGGAGAuuAGud   0 158ACuAAUCUCcACUUcAUCCd   0, 1 TsdT TsdT 160 (NH2C6)GfgAfuGfaAfgUfgGfa 101159 pasCfuAfaUfcUfcCfaCfuUfcAf  97 GfaUfuAfgUf(invdT) uCfc(invdT) 165(NH2C6)GfcAfaAfgGfcGfuGfc 103 166 puGfaGfuUfgGfcAfcGfcCfuUfu 103CfaAfcUfcAf(invdT) Gfc(invdT) 167 (NH2C6)GcAAAGGcGuGccAA  56 168UGAGUUGGcACGCCUUUGC  49 cucAdTsdT dTsdT 169 (NH2C6)GGAUfCfAUfCfUfCf  64170 GUfAAGACfUfUfGAGAUfGA  54 AAGUfCfUfUfACfdTsdT UfCfCfdTsdT 153GGAAUCuuAuAuuuGAUCcAs   0, 1 154 uuGGAUcAAAuAuAAGAuUC   0, 1 A cscsU 173(NH2C6)UfgAfcCfaCfaGfuCfg 102 174 pusUfuAfaUfcCfgAfcUfgUfgGf 102GfaUfuAfaAf(invdT) uCfa(invdT) 175 (NH2C6)uGAccAcAGucGGAu   0, 4 176puUuAAUCCGACUGUGGucA   0, 3 uAAAdTsdT dTsdT 175 (NH2C6)uGAccAcAGucGGAu  6 177 UUuAAUCCGACUGUGGUcA   3 uAAAdTsdT dTsdT

CONCLUSIONS

A. RNA strands containing at least one 2′-OH nucleotide (e.g. bothstrands of SEQ ID NO pair 157/158) are rapidly degraded via a cyclicpentavalent intermediate, leading to 2′-3′ cyclic phosphates at the5′-cleavage product. The formation of the pentavalent intermediate canbe inhibited using nucleotides lacking a 2′-OH group, like e.g.2′-deoxy, 2′-OMe or 2′-F.B. Additionally, RNA is degraded via a 5′-exonucleolytic pathway, thatis independent from the 2′-modification on the 5′-terminal nucleotides.This degradation pathway can be inhibited using 5′-terminalnon-nucleotide moieties, like e.g. a C6-aminolinker (e.g. SEQ ID NO 160in SEQ ID NO pair 160/159 or SEQ ID NO 165 in SEQ ID NO pair 165/166) ora phosphorothioate at the first internucleotide linkage (e.g. SEQ ID NO160 in SEQ ID NO pair 160/159).C. A 5′-phosphate group slows down the exonucleolytic cleavage kinetics,but can not fully block the degradation starting at this end (e.g. SEQID NO 160 in SEQ ID NO pair 160/159). This is most probably due to thecleavage of the 5′-phosphate by either phosphatases or by an inherentphosphatase activity of the DNase II enzyme.D. The best protection for RNA strands was achieved witholigonucleotides containing no 2′-OH nucleotide within the strand,starting with a 2′-OMe nucleotide at the 5′-end connected by aphosphorothioate linkage to the second nucleotide (e.g. SEQ ID NO 173 inSEQ ID NO pair 173/174). Other terminal non-2′-OH nucleotides alsoprotect against the 5′-exo degradation, but to a lower extent comparedto the 2′-OMe modification (refer to Table 9)

Example 42 In Vivo Knock Down Activity of siRNAs Devoid of 2′-OHResidues

In vivo experiments were conducted with mice injected with Factor VII(FVII)-targeting siRNAs (SEQ ID NO pairs 179/166 and 180/168, see Table5) co-administered with DPC-GalNac.

TABLE 5 a Sequences of siRNAs for in vivo experiment. Key: lowercase letters a, c, g, u, are 2′-O-Methyl nucleotides;Upper case letters A, C, G, U followed by “f” indicatesa 2′-fluoro nucleotide. Lower case “p” indicates a 5′-phosphate. (invdT) represents an inverted deoxythimidine(3′-3′-linked). A phosphorothioate linkages is symbolizedwith a lower case “s”. dT is deoxythimidine. (NHC6) isthe aminohexyl linker incorporated at the 5′-end of thesense strand. GalNAc refers to the structure in formula (IV). SEQ IDSEQ ID NO pair NOs Sequence 5′->3′ 179/166 179GalNAc-(NHC6)- GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 166puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 180/168 180GalNAc-(NHC6)-GcAAAGGcGuGccAAcucAdTsdT 168 UGAGUUGGcACGCCUUUGCdTsdT

A FVII siRNA with an alternating 2′-OMe/2′-F pattern on sense andantisense strand was generated with a 5′-terminal 2′-OMe nucleotide onthe antisense and a 5′-terminal 2′-F strand on the sense strand. Bothstrands are protected by an inv(dT) at the 3′-terminal overhang. Theantisense strand was bearing a 5′-phosphate group to maintain activityof the siRNA. The sense strand was conjugated to a GalNAc-palmitoylconstruct at its 5′ end for targeting to hepatocytes by theasialyloglycoprotein-receptor. siRNA (2.5 mg/kg) was co-administeredwith GalNAc-targeted PBAVE (15 mg/kg) in mice.

FVII mRNA measurements were done from liver homogenates using QuantiGene1.0 branched DNA (bDNA) Assay Kit (Panomics, Fremont, Calif., USA,Cat-No: QG0004).

At necropsy 1-2 g liver tissue was snap frozen in liquid nitrogen.Frozen tissue was powderized with mortar and pistil on dry ice. 15-25 mgof tissue was transferred to a chilled 1.5 mL reaction tube, 1 mL 1:3Lysis Mixture prediluted in MilliQ water and 3.3 μL Proteinase K (50μg/μL) was added and tissue was lysed by several seconds ultrasoundsonication at 30-50% power (HD2070, Bandelin, Berlin, Germany). Lysateswere stored at −80° C. until analysis. For mRNA analysis lysate wasthawed and digested with Proteinase K for 15 min at 1000 rpm in athermomixer at 65° C. (Thermomixer comfort, Eppendorf, Hamburg,Germany). FVII and GAPDH mRNA levels were determined using QuantiGene1.0 bDNA Assay Kit reagents according to the manufacturer'srecommendations. FVII mRNA expression was analyzed using 20 μL lysateand a mouse FVII probe set. GAPDH mRNA expression was analysed using 40μL lysate and rattus norwegicus probe sets shown to be cross-react withmice (sequences of probesets see above). As assay readout thechemiluminescence signal at end of the assay was measured in a Victor 2Light luminescence counter (Perkin Elmer, Wiesbaden, Germany) asrelative light units (RLU). The signal for FVII mRNA was divided bysignal for GAPDH mRNA from the same lysate Values are reported as FVIImRNA expression normalized to GAPDH.

Results demonstrate a 79% FVII mRNA knock down at 48 hours post dosingafter administration of SEQ ID NO pair 179/166. In contrast, the 2′-OHnucleotide bearing siRNA SEQ ID NO pair 180/168 showed no significantknock down (<25%), as shown in Table 5.

TABLE 5 b Results of in vivo knockdown studies Time SEQ ID NO pair179/166 SEQ ID NO pair 180/168 [hour] Remaining mRNA [%] Remaining mRNA[%] 1 84 92 6 83 88 24 53 100 48 21 76

Example 43 Tissue Distribution of siRNAs Devoid of 2′-OH Residues

The siRNA concentration in the liver tissue samples was determined usinga proprietary oligonucleotide detection method as described inWO2010043512. Briefly, the siRNA quantification is based on thehybridization of a fluorescently (Atto-425) labeled PNA-probe(Atto425-OO-GCAAAGGCGTGCCAACT (SEQ ID NO: 270), obtained from PanageneInc, Korea) complementary to the antisense strand of the siRNA duplex,followed by AEX-HPLC based separation. Quantification was done byfluorescence detection against an external calibration curve that wasgenerated from a dilution series of the two FVII siRNA used in the invivo experiment (see example 42). For plasma samples between 0.2 to 2 μLand for tissue ˜1 mg aliquots were injected onto the HPLC system.

Liver tissue analysis of the stabilized siRNA lacking 2′-OH nucleotideshowed high concentrations of intact antisense strand in the liver inthe ug/g range, but ˜95% was present in the 5′-dephosphorylated inactiveform (see table IR.04). The resulting RNA with a terminal 2′-OMenucleotide is not prone for rephosphorylation in the cytoplasm by thephosphokinase hClp1 (see below). In contrast, the antisense strand ofthe 2′-OH containing siRNA was completely degraded in the tissue withinthe first 6 hours post dosing.

TABLE 6 Liver tissue analysis of the stabilized siRNA containing no2′-OH nucleotide SEQ ID NO pair 181/186 SEQ ID NO pair 181/185 Time inLiver [ng/g] in Liver [ng/g] [hour] −5′-Phosphat +5′-Phosphat−5′-Phosphat +5′-Phosphat 1 873 171 9 BDL 6 1351 106  BDL* BDL 24 104365 BDL BDL 48 1062 66 BDL BDL *BDL = below detection limit

Example 44 In Vitro Knock Down Activity of siRNAs with Optimized 5′-Ends

An additional in vitro screen for FVII siRNAs was conducted in order toidentify siRNAs that may be intracellularly (re-)phosphorylated at theantisense's 5′-end to result in the RNAi-competent species All siRNAsfrom this screen are shown in Table 7. The alternating 2′-OMe/2′-Fmodification pattern was identical to the 1^(st) generation design(without any 2′-OH residues) with exception of various modifications atthe first two nucleotides at the 5′-end of the antisense strand. The two5′-terminal nucleotides of the antisense strand were generated as 2′-For 2′-deoxy modified nucleotides in various combinations with andwithout an additional 5′-phosphate or 5′-phosphothioate. All siRNAs werescreened in dose response (24 nM to 0.00037 nM in 4fold dilutions) forknock down activity after transfection of primary mouse hepatocytes(30000 cells per well; 96 well plate formate) using Lipofectamine 2000according to the manufacturer's instructions. Two siRNAs were comparableactive to the parental duplex (SEQ ID NO pair 182/168); comparableactive siRNAs: SEQ ID NO pairs 181/186 and 181/185) in terms of IC₅₀values, one with a 5′-terminal 2′-F and a phosphate group and one withtwo 5′-terminal 2′-deoxy nucleotides and a 5′-phosphorothioate (seeTable 7 for IC50 values). Both of them are ˜5-6-fold more activecompared to the siRNA (SEQ ID NO pair 181/166) used in the first animalexperiment with the terminal 2′-OMe nucleotide.

TABLE 7 IC 50 values SEQ SEQ ID ID IC50 NO Sense strand sequence (5′-3′)NO Sense strand sequence (5′-3′) (nM) 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 185pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.17 182 GcAAAGGcGuGccAAcucAdTsdT168 UGAGUUGGcACGCCUUUGCdTsdT 0.228 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 186psdTdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.228 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 187psdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.554 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 188pdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.631 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 189pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.702 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 190pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 0.749 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 166puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 1.002 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 191psUfGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 1.185 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 192UfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 2.257 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 193psUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 2.428 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 194psdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT 3.208 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 195usGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 3.974 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 196uGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 4.235 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 197dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 4.235 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 198psdTGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 4.704 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 199dTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 5.341 183(Chol)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invd 190pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 9.771

Example 45 In Vitro 5′-Phosphorylation of siRNAs with Optimized5′-Termini

All siRNAs without a 5′-phosphate or 5′-phosphorothioate listed in Table7 were assessed for phosphorylation by hClp1 in a HeLa S100 cellextract.

5′-phosphorylation s was analyzed from S100 HeLa extracts as describedby Weitzer and Martinez (S. Weitzer and J. Martinez. hClp1: a novelkinase revitalizes RNA metabolism. Cell Cycle 6 (17):2133-2137, 2007).Directly after incubation of 1 μM siRNAs in the S100 HeLa extractcontaining 5 mM ATP, the solution was analyzed by either IP-RP-HPLC orAEX-HPLC under denaturing conditions by injection of 5 μL samplesolution:

-   A. IP-RP-HPLC was done employing a Waters XBridge C₁₈ column (2.5×50    mm, 2.5 μm particle size) at 65° C. column temperature. Gradient    elution was performed using 100 mM hexafluoroisopropanol (HFIP) and    16 mM triethylamine in 1% methanol as eluent A and composition A in    95% methanol as eluent B. A gradient from 1% B to 18% B in 30    minutes was employed.-   B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4×250 mm)    at 50° C. using a 20 mM phosphate buffer containing 10% ACN at    pH=11. Eluent B contained 1 M NaBr in eluent A. A gradient from 25    to 62% B in 18 minutes was employed.

The ratio of 5′-phosphorylation is calculated for each strand of a siRNAfrom the UV trace at 260 nm using the following equitation (PA is peakarea):%_((5′-phosphorylation))=100*PA_([5′-phosphorylated strand])/(PA_([5′-phosphorylated strand])+PA_([parent strand]))

In Table 8 is shown, that the antisense strand of an siRNA cannot be5′-phosphorylated, when a 2′-OMe nucleotide is located at the5′-terminus (SEQ ID NO pair 181/196 and SEQ ID NO pair 181/195). Incontrast the antisense strand is susceptible to 5′-phosphorylation, whena 2′-F, 2′-deoxy or 2′-OH nucleotide is incorporated at the 5′-terminus(SEQ ID NO pair 181/195, SEQ ID NO pair 181/192, SEQ ID NO pair 181/197,SEQ ID NO pair 181/199 and SEQ ID NO pair 182/168). The two siRNAs, thatwere comparably active in the in vitro assay as the parental SEQ ID NOpair 182/168 (SEQ ID NO pair 181/186 and 181/185), are susceptible to5′-phosphorylation once the synthetically introduced 5′-phosphate/5′-PTOgroup is cleaved in vivo, eg. by phosphatases.

TABLE 8Percentage of 5′-phosphorylated strand after 4 hours incubation in S100HeLa cell extract. Key: lower case letters a, c, g, u, are 2′-O-Methylnucleotides; Upper case letters A, C, G, U followed by “f” indicates a2′-fluoro nucleotide.(invdT) represents an inverted deoxythimidine(3′-3′-linked). A phosphorothioate linkages is symbolized with a lowercase “s”. dT is deoxythimidine. SEQ sense SEQ antisense IDSense strand sequence 5′P ID Antisense strand sequence  5′P NO (5′-3′)[%] NO (5′-3′) [%] 181 GfcAfaAfgGfcGfuGfcCfaAfcUfcAf 52 196uGfaGfuUfgGfcAfcGfcCfuUfuGfc   0 (invdT) (invdT) 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf 53 195 usGfaGfuUfgGfcAfcGfcCfuUfuGfc   0(invdT) (invdT) 181 GfcAfaAfgGfcGfuGfcCfaAfcUfcAf 44 192UfsGfaGfuUfgGfcAfcGfcCfuUfuGfc  22 (invdT) (invdT) 181GfcAfaAfgGfcGfuGfcCfaAfcUfcAf 42 197 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc  22(invdT) (invdT) 181 GfcAfaAfgGfcGfuGfcCfaAfcUfcAf 47 199dTsdGaGfuUfgGfcAfcGfcCfuUfuGf  13 (invdT) c(invdT) 182GcAAAGGcGuGccAAcucAdTsdT 31 168 UGAGUUGGcACGCCUUUGCdTsd  42 T 184GfcAfaAfgGfcGfuGfcCfaAfcUfcA 22 168 UGAGUUGGcACGCCUUUGCdTsd 100 T

Example 46 In Vitro DNAse II-Stability of siRNAs with Optimized 5′ Ends

All antisense strands were screened for DNAse II stability as describedin example 41. The two antisense strands present in the siRNAs that werecomparable active to the parental duplex (SEQ ID NO 186 and SEQ ID NOpair 185 one with a 5′-terminal 2′-F and a phosphate group and one withtwo 5′-terminal 2′-deoxy nucleotides and a 5′-phosphorthioate are stabletowards DNAse II cleavage II (>70% intact strand after 20 hrincubation).

TABLE 9 In vitro stability of siRNAs towards DNase II after20 hours incubation Sense Antisense % SEQ ID SEQ ID intact NO NOSequence (5′-3′) strand 181 192 UfsGfaGfuUfgGfcAfcGfc70.CfuUfuGfc(invdT) 11 181 197 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)   0 181 199dTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)   0 181 193psUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 106 181 187psdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  96 181 194psdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 101 181 191psUfGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 100 181 198psdTGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  95 181 186psdTdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  99 181 185pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  71 181 189pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  74 181 188pdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)  64

Example 47 In Vivo Knock Down Activity of siRNAs with Optimized 5′ Ends

In order to evaluate if the in vitro improvement by optimized 5′-endstransfers to the in vivo situation, we conducted further mouseexperiments with GalNAc-palmitoyl conjugates of selected siRNAs (seeTable 10). SiRNAs were administered as under identical conditions asdescribed for the first mouse experiment (example 42, this patentapplication).

For measurement of FVII levels, plasma samples from mice were preparedby collecting blood (9 volumes) by submandibular bleeding intomicrocentrifuge tubes containing 0.109 mol/L sodium citrateanticoagulant (1 volume) following standard procedures. FVII activity inplasma was measured with a chromogenic method using a BIOPHEN VII kit(Hyphen BioMed/Aniara, Mason, Ohio) following manufacturer'srecommendations. Absorbance of colorimetric development was measuredusing a Tecan Safire2 microplate reader at 405 nm.

The siRNAs under investigation showed improved in vivo activity, fullycorrelating with the in vitro screening results. FVII activity in serumwas reduced by more than 80% for both siRNAs 48 hours post dosing,compared to 49% using the first generation DNase II stable siRNA design(see Table 10). This result clearly underscores the importance of a5′-terminal nucleotide on the antisense strand that can be effectivelyphosphorylated, in case phosphatases in vivo cleave the syntheticallygenerated 5′-phosphate or 5′-phosphothioate group. In case of a5′-terminal 2′-OMe nucleotide as used in the first design or describedin the literature as a more potent siRNA design based on in vitrocomparison with canonical siRNAs. (Allerson et al. J. Med Chem. 2005,48, 901-904), the cleavage of the synthetic phosphate in vivo would leadto a strong reduction in potency of the corresponding siRNA.

TABLE 10In vivo knockdown activity of siRNAs with optimized 5′ends. Key:lower case letters a, c, g, u, are 2′-O-Methyl nucleotides; Uppercase letters A, C, G, U followed by “f” indicates a 2′-fluoronucleotide. Lower case “p” indicates a 5′-phosphate. (invdT)represents an inverted deoxythimidine (3′-3′-linked). A phos-phorothioate linkages is symbolized with a lower case “s”.(NHC6)is the aminohexyl linker incorporated at the 5′-end of the sensestrand. GalNAc refers to the structure in formula (IV). % SEQ SEQremaining ID Sense strand sequence ID Antisense strand sequence FVII inNO (5′-3′) NO (5′-3′) serum 179 GalNAc-(NH2C6)- 166puGfaGfuUfgGfcAfcGfcCfuUfuGfc 27 GfcAfaAfgGfcGfuGfcCfaAfcUfc (invdT)Af(invdT) 179 GalNAc-(NH2C6)- 190 pusGfaGfuUfgGfcAfcGfcCfuUfuGfc 51GfcAfaAfgGfcGfuGfcCfaAfcUfc (invdT) Af(invdT) 179 GalNAc-(NH2C6)- 185pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc 17 GfcAfaAfgGfcGfuGfcCfaAfcUfc (invdT)Af(invdT) 179 GalNAc-(NH2C6)- 263 psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 13GfcAfaAfgGfcGfuGfcCfaAfcUfc (invdT) Af(invdT)

Example 48 In Vitro Knock Down Activity of siRNAs with Optimized 3′ Ends

To further increase activity of the DNase II stable siRNAs an SAR studyof the 3′-overhang was performed. Various combinations of invdT, dTinvdTor dTsdT on either the sense or the antisense strand 3′-overhang wereapplied to Aha1- and EGFP-targeting siRNAs (see Tables 11 and 12,respectively) and were pairwise compared for composition of both 3′endsin most potent siRNAs. All siRNAs were screened in dose response (24 nMto 0.00037 nM in 4-fold dilutions) for knock down activity aftertransfection of primary mouse hepatocytes (30000 cells/well; 96 wellplate format) using Lipofectamine2000 according to the manufacturer'sinstructions.

TABLE 11In vitro knock down activity of EGFP-targeting siRNAs with different 3′-ends.SEQ SEQ ID Sense strand sequence ID Antisense strand sequence IC50 NO(5′-3′) NO (5′-3′) (nM  45 GCUGGAGUUCGUGACCGCCdTdT  46GGCGGUCACGAACUCCAGCdTdT 1.0490 212 GcuGGAGuucGuGAccGccdTsdT 225GGCGGUcACGAACUCcAGCdTsdT #N/A 201 gcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)221 dGsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT) 0.4377 201gcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 214dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.1479 211gcUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT) 223dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT) 0.5833 203gcUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT 214dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.2166 204GfcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 224pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT) 0.9100 204GfcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 215pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.2241 207GfcUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT) 218pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT) 0.3474 206GfcUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT 215pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.2392 205GfscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 220GfsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT) 0.4251 205GfscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 216GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.2349 210GfscUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT) 222GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT) 0.5230 209GfscUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT 216GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.4937 200gscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 217pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT) 0.2643 200gscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT) 213pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.0936 208gscUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT) 219pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT) 0.3776 202gscUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT 213pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT 0.1569

TABLE 12In vitro knock down activity of Aha1-targeting siRNAs with different 3′-ends.SEQ SEQ ID Sense strand sequence ID Antisense strand sequence IC50 NO(5′-3′) NO (5′-3′) (nM 157 GGAuGAAGuGGAGAuuAGudTsdT 158ACuAAUCUCcACUUcAUCCdTsdT 0.094 234 GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)246 AfsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT) 0.081 234GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 240AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.036 233GfgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT) 239AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT) 0.034 236GfgAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT 240AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.040 231GfsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 241pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT) 0.037 231GfsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 267pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.030 229GfsgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT) 268pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT) 0.024 228GfsfAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT 267pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.021 232ggAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 245dAsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT) 0.060 232ggAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 238dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.030 237ggAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT) 244dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT)  0.045 230ggAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT 238dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.025 227gsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 243pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT) 0.045 227gsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 266pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.015 235gsgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT)  242pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT) 0.039 226gsgAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT 266pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 0.014

It was found, that siRNAs with 2 nucleotide dTsdT-overhangs on theantisense strand performed always better than those with a single invdToverhang at the antisense's 3′-end (while sense strands were the same).Further beneficial was the combination with a sense strand modified witha single invdT-overhang as 3′ overhang.

Example 49 In Vivo Knock Down Activity of siRNAs in Non-Human Primates

Preparation of DPCs and Dosing

DPCs were prepared by covalently attaching polymer “149 RAFT” to theindicated siRNA targeting coagulation Factor VII (siF7) at 4:1 wt:wtratio (polymer:siRNA) through a disulfide linkage and then modifying thepolymer-siRNA conjugate with a 2:1 wt:wt mixture of CDM-PEG:CDM-NAG at a7× wt:wt ratio (CDM:polymer). Cynomolgous monkeys were dosed with 1mg/kg DPC (polymer weight) and 0.25 mg/kg of the indicated siRNA. Oneanimal received DPC containing siF7 SEQ ID NO pair 151/152, two animalsreceived DPC containing siF7 SEQ ID NO pair 253/254), #1 and #2), andtwo animals received DPC containing SEQ ID NO pair 251/255, #1 and #2).F7 values were normalized to the average of the two pre-dose values.Animals receiving DPCs containing SEQ ID NO pair 253/254 or SEQ ID NOpair 251/255 had greater levels of F7 knockdown and longer PT than theanimal receiving SEQ ID NO pair 251/252.

DPC Injection Procedure

For each injection procedure, animals were given an IM injectioncontaining a combination of ketamine (up to 7 mg/kg) and dexmedetomidine(up to 0.03 mg/kg) and moved to a procedure room. In the procedure room,animals were placed on a water jacketed heating pad and the injectionsite was shaved and prepped with an antiseptic. An intravenous catheter(20 to 22 gauge) was inserted into a systemic vein (cephalic or smallsaphenous) and the DPC solution was infused (2 ml/kg) slowly over 1 to 2minutes. A pulse oximeter was used to monitor the heart rate and oxygensaturation during and immediately following the injection procedure.Each injection procedure took about 20 minutes to perform. Afterinjection the catheter was removed and gentle

pressure was applied to the venipuncture site. Animals were taken backto their cages and given an IM injection of the reversal drugatipamezole (antisedan) (0.10 to 0.15 mg/kg). Animals were monitoreduntil they regained normal activity.

Blood Collection Procedure

Blood samples (1-5 ml) were obtained for the measurement of geneinhibition (F7 activity, coagulation time), blood chemistries, andmarkers of liver damage (CBC, chemistry panel, ALT, cytokines,complement). For these blood collection procedures, animals were givenan IM injection containing a combination of ketamine (up to 7 mg/kg) anddexmedetomidine (up to 0.03 mg/kg). Once sedated, animals were moved onto a portable procedure table and a 22 gauge needle and syringe wereused to collect blood from the femoral vein. Immediately after the bloodcollection, pressure was applied to the venipuncture site and the bloodwas divided into the appropriate sample tubes for each blood test.Animals were then given an IM injection of the reversal drug atipamezole(antisedan) (0.10 to 0.15 mg/kg) and returned to their cage. No morethan 20% of total blood volume was drawn in any 30-day period (estimatedblood volume=60 ml/kg). Each blood collection procedure took about 10minutes to perform.

Factor VII (F7) Activity Measurements

Blood samples from non-human primates were prepared by filling serumseparator tubes with whole blood and allowing the blood to clot at roomtemperature for at least 20 minutes. After clotting, blood tubes werecentrifuged for 3 minutes at 9000 rpm, aliquoted into eppendorf tubes,and stored at −20° C. until assayed. F7 activity in serum was measuredwith a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara,Mason, Ohio) following manufacturer's recommendations. Absorbance ofcolorimetric development was measured using a Tecan Safire2 microplatereader at 405 nm.

Coagulation Tests (Protime, Partial Protime and Fibrinogen)

Blood samples from non-human primates were prepared by completelyfilling sodium citrate tubes (BD Vacutainer) with whole blood and gentlymixing to prevent clot formation. Tubes were transported to a clinicaltesting lab within one hour and coagulation assays were performed within4 hours from the time of collection.

TABLE 13 FVII SiRNA used for NHP experiment: Key: lower case lettersa, c, g, u, are 2′-O-Methyl nucleotides; Upper case lettersA, C, G, U followed by “f” indicates a 2′-fluoro nucleotide.Lower case “p” indicates a 5′-phosphate. (invdT) representsan inverted deoxythimidine (3′-3′-linked). A phosphorothioatelinkages is symbolized with a lower case “s”. dT is deoxythimidine. SEQSEQ ID Sense strand sequence ID Antisense strand sequence NO (5′-3′) NO(5′-3′) 251 (NH2C6)GfuUfgGfuGfaAfuGfgAfgCfuCf 252pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfc aGf(invdT) (invdT) 253(NH2C6)GfgUfcCfuGfuUfgUfuGfgUfgAf 254 pAfsUfuCfaCfcAfaCfaAfcAfgGfaCfcdaUf(invdT) TsdT 251 (NH2C6)GfuUfgGfuGfaAfuGfgAfgCfuCf 255pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfcd aGf(invdT) TsdT

Changing from an single nucleotide (invdT)-3′-overhang on both strandsto an asymmetric siRNA design with a 3′-(invdT) overhang on the sensestrand and a dTsdT overhang on the antisense strand, but otherwiseconstant modification pattern lead to a more pronounced serum FVIIreduction and a significantly prolonged duration of this effect innon-human primates (see FIG. 6a ). This observation is supported by anexpected biologic consequence, namely a more pronounced effect on theprothrombin time corresponding to the extent of Factor 7 reduction (seeFIG. 6b ).

Example 50 In Vivo Knock Down Activity of siRNAs with Cleavable RNALinkers

In Table 14 the in vivo efficacy based on FVII protein inhibition inserum was compared using cholesterol or the GalNAc-palmitoyl siRNAconjugate in the same sequence context in mice. The in vivo experimentwas conducted as described in example 42. FVII inhibition was stronglydecreased for the cholesterol conjugated siRNAs containing no 2′-OHnucleotide compared to the GalNAc-palmitoyl conjugated counterparts (SEQID NO pair 179/166 vs. 179/190, SEQ ID NO pair 257/264 vs. SEQ ID NOpair 179/262, SEQ ID NO pair 257/263 vs. SEQ ID NO pair 179/163 and SEQID NO pair 257/166 vs. (SEQ ID NO pair 179/166). In contrast for a 2′-OHcontaining siRNA the cholesterol conjugate lead to higher FVIIinhibition compared to the GalNAc-palmitoyl derivative (SEQ ID NO pair180/168 vs. SEQ ID NO pair 258/168).

The small molecule ligands GalNAc-palmitoyl and cholesterol used in thedescribed in vivo experiment are connected to the siRNA via anon-cleavable linker to the 5′-end of the sense strand. In case thesense strand exhibit 2′-OH nucleotides the ligand is still cleavable bynucleases (e.g. DNase II in the endosomal or lysosomal compartment). Thecleavage reaction releases the free siRNA that is then released into thecytoplasm by the endosomal perturbing activity of the delivery polymer.

For siRNAs lacking a 2′-OH nucleotide in the sense strand, the ligandsare stably connected to the duplex, as no enzymatic(nuclease/protease/esterase etc.) or chemical mechanism triggers thecleavage of the ligand. Therefore, fully stable cholesterol conjugatedsiRNA can be trapped in cell membranes due to the membrane interactionof the lipophilic cholesterol ligand. Even high concentrations of thesiRNA in the tissue is not sufficient for effective release of the siRNAinto in the cytoplasm. In contrast, the less lipophilic GalNAc-palmitoylconjugated siRNA can be released into the cytoplasm, due to a lesspronounced interaction with cell membranes. For this reason a stable,non-cleavable GalNAc-palmitoyl siRNA conjugate is more efficaciouscompared to a cholesterol conjugated to the same siRNA.

Developing cleavable linker constructs would help to circumvent theissue of membrane trapping for stably conjugated cholesterol siRNA.Using disulfide linker chemistry is described as an attractivepossibility to introduce a defined cleavage site but cleavage is mostprobably restricted to the reducing environment of specific organelleswithin the cell (PNAS, 2006, 103, 13872). As cleavage is expected to beslow in the endosomal/lysosomal compartment most of thecholesterol-disulfide conjugated siRNA can still be trapped in membranesas described for the non-cleavable cholesterol conjugates.

TABLE 14 SEQ % FVII SEQ ID ID activity activity Conjugate NO pair NOSequence 5′->3′ in serum GalNA

179/166 179 GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 27 166puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 179/190 179GalNAc-NH2C6-GfcAfaAfgGfcGfuGFcCfaAfcUfcAf(invdT) 51 190pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 179/262 179GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 17 262pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 179/263 179GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 13 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 180/168 180GalNAc-NH2C6-GcAAAGGcGuGccAAcucAdTsdT 86 168 UGAGUUGGcACGCCUUUGCdTsdTCholesterol 257/166 257Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 48 166puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 257/190 257Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 93 190pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 257/264 257Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 63 264pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 257/263 257Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 41 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT) 258/168 258Cholesterol-NH2C6-GcAAAGGcGuGccAAcucAdTsdT 50 168UGAGUUGGcACGCCUUUGCdTsdT

In addition to the well-established disulfide cleavable linker chemistryanother possibility is the generation of defined cleavage sites by using2′-OH nucleotides at certain positions. Introduction of 2′-OHnucleotides at selective positions is a new approach to achieve cleavageof the conjugates from RNA strands. The 2′-OH nucleotides can either beimplemented by adding single stranded overhangs with at least one2′-OH-nucleotide at the 3′- or 5′-end of the RNA strand or by using2′-OH nucleotides within the duplex region of an siRNA. The enzymaticactivity of nucleases present in the endosome/lysosome cleavesselectively at this positions. In a first design the cholesterol wasconnected to the sense strand via a single stranded overhang containing3 2′-OH nucleotides (AUC) at the 5′-terminus.

Cholesterol conjugated siRNAs comparing various cleavable linkerchemistries are shown in Table 15. All siRNAs have the identicalsequence context, just the linker chemistry was altered. Cholesterol wasconnected to the sense strand via single stranded overhang comprised ofa three 2′-OH nucleotides (AUC) to the 5′-terminus. When co-administeredwith a delivery polymer this siRNA (SEQ ID NO pair 260/263) lead to 77%FVII down modulation in serum in mice, compared to only 60% when usingthe identical siRNA with a stably attached cholesterol (SEQ ID NO pair257/263). The same siRNA with a cholesterol conjugated via a linkeraccording to formula Ia to the 5′-terminus of the sense strand (SEQ IDNO pair 261/263) lead to 93% FVII activity reduction in serum. Allresults were achieved by co-administration of 15 mg/kg of a deliverypolymer with 2.5 mg/kg of the cholesterol conjugated siRNA in mice.

TABLE 15 In vivo comparison of various linker chemistries forcholesterol conjugated siRNAs SEQ SEQ % FVII ID Sense strand sequence IDAntisense strand sequence activity NO (5′-3′) NO (5′-3′) in serum 257Chol-(NH2C6)- 263 psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 40GfcAfaAfgGfcGfuGfcCfaAfcUfcAf (invdT) (invdT) 259 Chol-C6SSC6- 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 22 GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) (invdT) 260 Chol-AUC- 263 psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 23GfcAfaAfgGfcGfuGfcCfaAfcUfcAf (invdT) (invdT) 261 Chol-Cathepsin- 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 7 GfcAfaAfgGfcGfuGfcCfaAfcUfcAf (invdT)(invdT)

These results indicate, that the use of a cleavable linker improves thein vivo potency of siRNAs containing no 2′-OH nucleotide. The cleavablelinker can either comprised of 2′-OH containing nucleotides, adi-peptide cleavage motive or a disulfide linker chemistry. Allcleavable linker constructs improve the in vivo potency in aco-administration setup of a cholesterol conjugated siRNAs with a slowendosomal release delivery polymer.

Example 51 In Vitro Serum Stability of siRNAs with Cleavable Linkers

The stability of the cleavable linker was evaluated in an in vitrostability assay. The cholesterol conjugated sense strands were incubatedin 90% mouse serum at 37° C. for various time points. The incubationreaction was stopped by addition of proteinase K in a sodium dodecylsulfate (SDS) containing buffer—The treatment degrades all proteins andenzymes without interfering with the RNA strand integrity. 25 μL of thissolution was directly injected onto a AEX-HPLC system connected to a UVdetector at 260 nm. AEX-HPLC was performed on a Dionex DNA Pac200 column(4×250 mm) at 75° C. using a 20 mM Tris buffer containing 50% ACN atpH=8. 800 mM NaBr in eluent B serves as eluent salt. A gradient from 25to 62% B in 18 minutes was employed.

The cholesterol containing single stranded RNA elutes from the HPLCcolumn as a broad peak at 260 nm After cleavage of the cholesterol sharpsymmetric peaks is observed at lower retention time. Cleavage rate ofcholesterol was determined by the following equitation (PA=Peak Area):%_((free RNA))=100*PA_([free RNA])/(PA_([free RNA])+PA_([cholesterol conjugated RNA]))

In vitro it was shown, that the 3 nt nucleotide (AUC)-overhang isquantitatively cleaved in less than 1 hour in 90% mouse serum. Thecleavage occurs 3′ to the two pyrimidine nucleotides in the overhang,leading to two distinct cleavage metabolites (peak areas of metaboliteswere summarized for data evaluation). In contrast, the di-peptidecontaining linker according to formula Ia, the disulfide and the stablylinked cholesterol are fully stable in mouse serum.

Example 52 Tissue Distribution of siRNAs with Cleavable Linkers

The siRNA concentration in the liver tissue samples was determined usinga proprietary oligonucleotide detection method as described inWO2010043512. Briefly, the siRNA quantification is based on thehybridization of a fluorescently (Atto-425) labeled PNA-probe(Atto425-OO-TGAGTTGGCACGCCTTT (SEQ ID NO: 269) obtained from PanageneInc, Korea) complementary to the sense strand of the siRNA duplex,followed by AEX-HPLC based separation. Quantification was done byfluorescence detection against an external calibration curve that wasgenerated from a dilution series of the two FVII siRNA used in the invivo experiment (see example 42). For plasma samples between 0.2 to 2 μLand for tissue ˜1 mg aliquots were injected onto the HPLC system.

In Table 16 results from liver tissue analysis are shown. When analyzingthe siRNA content it was found, that the sense strand that is present inliver tissue, is quantitatively cleaved from cholesterol when usingeither the di-peptide linker motive or the 3 nt 5′-overhang with theunmodified linker sequence AUC. In contrast, only 15% of the disulfidelinked siRNA that is present in the liver is cleaved from cholesterolwithin the first 48 hours post dosing and nothing of the stably attachedcholesterol is cleaved from the siRNA.

When comparing the absolute amounts of cholesterol-free siRNA in livertissue similar amounts were found for the disulfide linker and for theRNA AUC-linker, nicely correlating with equal FVII serum activity 48hours post dosing The lower FVII activity achieved with the di-peptidelinked cholesterol siRNA fully correlates with the higher absoluteamount of the cleaved cholesterol-free siRNA.

The total amount of cholesterol siRNA conjugate equipped with an(AUC)-linker on the sense strand delivered into the liver is ˜6-foldlower as compared to the stably or the disulfide attached cholesteroland ˜3-fold lower compared to the di-peptide conjugated cholesterolsiRNA. The reduced tissue presence can be attributed to the fact thatthe AUC-linker is not only a substrate for intracellular nucleases, butalso for nucleases present in circulation as shown in the in vitroincubation with mouse serum. When the cholesterol ligand is cleaved fromthe siRNA already in circulation the resulting siRNA is prone to renalclearance and is rapidly excreted into urine without delivery intotissue.

TABLE 16 Total % sense siRNA cleaved SEQ SEQ in ligand IDSense strand sequence ID Antisense strand sequence Liver in NO (5′-3′)NO (5′-3′) [ng/g] liver 257 Chol-(NH2C6)- 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 5837 0 GfcAfaAfgGfcGfuGfcCfaAfeUf(invdT) cAf(invdT) 259 Chol-C6SSC6- 263 psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc4357 14.8 GfcAfaAfgGfcGfuGfcCfaAfcUf (invdT) cAf(invdT) 260 Chol-AUC-263 psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc  912 96.1GfcAfaAfgGfcGfuGfcCfaAfcUf (invdT) cAf(invdT) 261 Chol-Cathepsin- 263psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc 2760 99.8 GfcAfaAfgGfcGfuGfcCfaAfcUf(invdT) cAf(invdT)In the following tables siRNAs used in the examples are summarized:

TABLE 17 Core sequences SEQ SEQ ID Sense strand ID Antisense strand NOsequence (5′-3′) NO sequence (5′-3′)   1 ACAUGAAGCAGCACGACUU   2AAGUCGUGCUGCUUCAUGU   3 GCCCGACAACCACUACCUG   4 CAGGUAGUGGUUGUCGGGC   5CGAGAAGCGCGAUCACAUG   6 CAUGUGAUCGCGCUUCUCG   7 AUAUCAUGGCCGACAAGCA   8UGCUUGUCGGCCAUGAUAU   9 ACAAGCUGGAGUACAACUA  10 UAGUUGUACUCCAGCUUGU  11GCAGCUCGCCGACCACUAC  12 GUAGUGGUCGGCGAGCUGC  13 CGUCCAGGAGCGCACCAUC  14GAUGGUGCGCUCCUGGACG  15 GCUGGAGUUCGUGACCGCC  16 GGCGGUCACGAACUCCAGC  17CCACCCUGACCUACGGCGU  18 ACGCCGUAGGUCAGGGUGG  19 CGACUUCAAGGAGGACGGC  20GCCGUCCUCCUUGAAGUCG  21 UUCAAGAUCCGCCACAACA  22 UGUUGUGGCGGAUCUUGAA  23GGCAACUACAAGACCCGCG  24 CGCGGGUCUUGUAGUUGCC  25 CCGGCAAGCUGCCCGUGCC  26GGCACGGGCAGCUUGCCGG  27 UGCCCAUCCUGGUCGAGCU  28 AGCUCGACCAGGAUGGGCA  29CAAGUUCAGCGUGUCCGGC  30 GCCGGACACGCUGAACUUG 151 GGAAUCUUAUAUUUGAUCCAA152 UUGGAUCAAAUAUAAGAUUCCCU 155 GGAUGAAGUGGAGAUUAGU 156ACUAAUCUCCACUUCAUCC 161 GCAAAGGCGUGCCAACUCA 162 UGAGUUGGCACGCCUUUGC 161GCAAAGGCGUGCCAACUCA 178 TGAGUUGGCACGCCUUUGC 161 GCAAAGGCGUGCCAACUCA 256TGAGUUGGCACGCCUUUGC 163 GGAUCAUCUCAAGUCUUAC 164 GUAAGACUUGAGAUGAUCC 171UGACCACAGUCGGAUUAAA 172 UUUAAUCCGACUGUGGUCA 247 GUUGGUGAAUGGAGCUCAG 248CUGAGCUCCAUUCACCAAC 249 GGUCCUGUUGUUGGUGAAU 250 AUUCACCAACAACAGGACC

TABLE 18 Mapping of core sequences and modified sequence Core sequencesSense Antisense Modified sequences SEQ strand SEQ strand SEQ SEQ IDsequence  ID sequence ID Sense strand ID Antisense strand NO (5′-3′) NO(5′-3′) NO sequence (5′-3′) NO sequence (5′-3′)   1 ACAUGAAGC   2AAGUCGUGC  31 ACAUGAAGCAG  32 AAGUCGUGCUGCU AGCACGACU UGCUUCAUGCACGACUUdTdT UCAUGUdTdT U U   1 ACAUGAAGC   2 AAGUCGUGC  61AfcAfuGfaAfgCfa  62 paAfgUfcGfuGfcUfg AGCACGACU UGCUUCAUGGfcAfcGfaCfuUfd CfuUfcAfuGfudTsdT U U TsdT   1 ACAUGAAGC   2 AAGUCGUGC 91 dAcdAudGadAgdC  92 padAgdTcdGudGcdTg AGCACGACU UGCUUCAUGadGcdAcdGadCud dCudTcdAudGudTsd U U TdTsdT T   1 ACAUGAAGC   2 AAGUCGUGC121 AfdCAfdTGfdAAf 122 pdAAfdGUfdCGfdTG AGCACGACU UGCUUCAUGdGCfdAGfdCAfdC fdCUfdGCfdTUfdCAf U U GfdACfdTUfdTsdT dTGfdTdTsdT   3GCCCGACAA   4 CAGGUAGUG  33 GCCCGACAACC  34 CAGGUAGUGGUU CCACUACCUGUUGUCGGG ACUACCUGdTdT GUCGGGCdTdT G C   3 GCCCGACAA   4 CAGGUAGUG  63GfcCfcGfaCfaAfcC  64 pcAfgGfuAfgUfgGfu CCACUACCU GUUGUCGGGfaCfuAfcCfuGfdTs UfgUfcGfgGfcdTsdT G C dT   3 GCCCGACAA   4 CAGGUAGUG 93 dGcdCcdGadCadA  94 pcdAgdGudAgdTgdG CCACUACCU GUUGUCGGGcdCadCudAcdCud udTgdTcdGgdGcdTsd G C GdTsdT T   3 GCCCGACAA   4CAGGUAGUG 123 GfdCCfdCGfdACf 124 pdCAfdGGfdTAfdGU CCACUACCU GUUGUCGGGdAAfdCCfdACfdT fdGGfdTUfdGUfdCG G C AfdCCfdTGfdTsdT fdGGfdCdTsdT   5CGAGAAGCG   6 CAUGUGAUC  35 CGAGAAGCGCG  36 CAUGUGAUCGCGC CGAUCACAUGCGCUUCUC AUCACAUGdTdT UUCUCGdTdT G G   5 CGAGAAGCG   6 CAUGUGAUC  65CfgAfgAfaGfcGfc  66 pcAfuGfuGfaUfcGfc CGAUCACAU GCGCUUCUCGfaUfcAfcAfuGfd GfcUfuCfuCfgdTsdT G G TsdT   5 CGAGAAGCG   6 CAUGUGAUC 95 dCgdAgdAadGcdG  96 pcdAudGudGadTcdG CGAUCACAU GCGCUUCUCcdGadTcdAcdAud cdGcdTudCudCgdTsd G G GdTsdT T   5 CGAGAAGCG   6CAUGUGAUC 125 CfdGAfdGAfdAGf 126 pdCAfdTGfdTGfdAU CGAUCACAU GCGCUUCUCdCGfdCGfdAUfdC fdCGfdCGfdCUfdTCf G G AfdCAfdTGfdTsdT dTCfdGdTsdT   7AUAUCAUGG   8 UGCUUGUCG  37 AUAUCAUGGCC  38 UGCUUGUCGGCCA CCGACAAGCGCCAUGAUA GACAAGCAdTdT UGAUAUdTdT A U   7 AUAUCAUGG   8 UGCUUGUCG  67AfuAfuCfaUfgGfc  68 puGfcUfuGfuCfgGfc CCGACAAGC GCCAUGAUACfgAfcAfaGfcAfd CfaUfgAfuAfudTsdT A U TsdT   7 AUAUCAUGG   8 UGCUUGUCG 97 dAudAudCadTgdG  98 pudGcdTudGudCgdG CCGACAAGC GCCAUGAUAcdCgdAcdAadGcd cdCadTgdAudAudTsd A U AdTsdT T   7 AUAUCAUGG   8UGCUUGUCG 127 AfdTAfdTCfdAUf 128 pdTGfdCUfdTGfdTCf CCGACAAGC GCCAUGAUAdGGfdCCfdGAfdC dGGfdCCfdAUfdGAf A U AfdAGfdCAfdTsd dTAfdTdTsdT T   9ACAAGCUGG  10 UAGUUGUAC  39 ACAAGCUGGAG  40 UAGUUGUACUCCA AGUACAACUUCCAGCUUG UACAACUAdTdT GCUUGUdTdT A U   9 ACAAGCUGG  10 UAGUUGUAC  69AfcAfaGfcUfgGfa  70 puAfgUfuGfuAfcUfc AGUACAACU UCCAGCUUGGfuAfcAfaCfuAfd CfaGfcUfuGfudTsdT A U TsdT   9 ACAAGCUGG  10 UAGUUGUAC 99 dAcdAadGcdTgdG 100 pudAgdTudGudAcdT AGUACAACU UCCAGCUUGadGudAcdAadCud cdCadGcdTudGudTsd A U AdTsdT T   9 ACAAGCUGG  10UAGUUGUAC 129 AfdCAfdAGfdCUf 130 pdTAfdGUfdTGfdTA AGUACAACU UCCAGCUUGdGGfdAGfdTAfdC fdCUfdCCfdAGfdCUf A U AfdACfdTAfdTsdT dTGfdTdTsdT  11GCAGCUCGC  12 GUAGUGGUC  41 GCAGCUCGCCG  42 GUAGUGGUCGGC CGACCACUAGGCGAGCUG ACCACUACdTdT GAGCUGCdTdT C C  11 GCAGCUCGC  12 GUAGUGGUC  71GfcAfgCfuCfgCfc  27 pgUfaGfuGfgUfcGfg CGACCACUA GGCGAGCUGGfaCfcAfcUfaCfdT CfgAfgCfuGfcdTsdT C C sdT  11 GCAGCUCGC  12 GUAGUGGUC101 dGcdAgdCudCgdC 102 pgdTadGudGgdTcdG CGACCACUA GGCGAGCUGcdGadCcdAcdTad gdCgdAgdCudGcdTsd C C CdTsdT T  11 GCAGCUCGC  12GUAGUGGUC 131 GfdCAfdGCfdTCf 132 pdGUfdAGfdTGfdGU CGACCACUA GGCGAGCUGdGCfdCGfdACfdC fdCGfdGCfdGAfdGC C C AfdCUfdACfdTsd fdTGfdCdTsdT T  13CGUCCAGGA  14 GAUGGUGCG  43 CGUCCAGGAGC  44 GAUGGUGCGCUCC GCGCACCAUCUCCUGGAC GCACCAUCdTdT UGGACGdTdT C G  13 CGUCCAGGA  14 GAUGGUGCG  73CfgUfcCfaGfgAfg  74 pgAfuGfgUfgCfgCfu GCGCACCAU CUCCUGGACCfgCfaCfcAfuCfdT CfcUfgGfaCfgdTsdT C G sdT  13 CGUCCAGGA  14 GAUGGUGCG103 dCgdTcdCadGgdA 104 pgdAudGgdTgdCgdC GCGCACCAU CUCCUGGACgdCgdCadCcdAud udCcdTgdGadCgdTsd C G CdTsdT T  13 CGUCCAGGA  14GAUGGUGCG 133 CfdGUfdCCfdAGf 134 pdGAfdTGfdGUfdGC GCGCACCAU CUCCUGGACdGAfdGCfdGCfdA fdGCfdTCfdCUfdGGf C G CfdCAfdTCfdTsdT dACfdGdTsdT  15GCUGGAGUU  16 GGCGGUCAC  45 GCUGGAGUUCG  46 GGCGGUCACGAAC CGUGACCGCGAACUCCAG UGACCGCCdTdT UCCAGCdTdT C C  15 GCUGGAGUU  16 GGCGGUCAC  75GfcUfgGfaGfuUfc  76 pgGfcGfgUfcAfcGfa CGUGACCGC GAACUCCAGGfuGfaCfcGfcCfd AfcUfcCfaGfcdTsdT C C TsdT  15 GCUGGAGUU  16 GGCGGUCAC105 dGcdTgdGadGudT 106 pgdGcdGgdTcdAcdG CGUGACCGC GAACUCCAGcdGudGadCcdGcd adAcdTcdCadGcdTsd C C CdTsdT T  15 GCUGGAGUU  16GGCGGUCAC 135 GfdCUfdGGfdAGf 136 pdGGfdCGfdGUfdCA CGUGACCGC GAACUCCAGdTUfdCGfdTGfdA fdCGfdAAfdCUfdCCf C C CfdCGfdCCfdTsdT dAGfdCdTsdT  15GCUGGAGUU  16 GGCGGUCAC 200 gscUfgGfaGfuUfc 213 pdGsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf aAfcUfcCfaGfcdTsdT C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 201 gcUfgGfaGfuUfcG 214 dGsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG fuGfaCfcGfcCf AfcUfcCfaGfcdTsdT C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 202 gscUfgGfaGfuUfc 213 pdGsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd aAfcUfcCfaGfcdTsdT C C TsdT  15GCUGGAGUU  16 GGCGGUCAC 203 gcUfgGfaGfuUfcG 214 dGsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG fuGfaCfcGfcCfdTs AfcUfcCfaGfcdTsdT C C dT  15GCUGGAGUU  16 GGCGGUCAC 204 GfcUfgGfaGfuUfc 215 pGfsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf aAfcUfcCfaGfcdTsdT C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 205 GfscUfgGfaGfuUfc 216 GfsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf AfcUfcCfaGfcdTsdT C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 206 GfcUfgGfaGfuUfc 215 pGfsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd aAfcUfcCfaGfcdTsdT C C TsdT  15GCUGGAGUU  16 GGCGGUCAC 200 gscUfgGfaGfuUfc 217 pdGsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf aAfcUfcCfaGfc(invdT) C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 207 GfcUfgGfaGfuUfc 218 pGfsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd aAfcUfcCfaGfcdT C C T(invdT) (invdT) 15 GCUGGAGUU  16 GGCGGUCAC 208 gscUfgGfaGfuUfc 219 pdGsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd aAfcUfcCfaGfcdT C C T(invdT) (invdT) 15 GCUGGAGUU  16 GGCGGUCAC 205 GfscUfgGfaGfuUfc 220 GfsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf AfcUfcCfaGfc(invdT) C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 201 gcUfgGfaGfuUfcG 221 dGsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG fuGfaCfcGfcCf AfcUfcCfaGfc(invdT) C C (invdT)  15GCUGGAGUU  16 GGCGGUCAC 209 GfscUfgGfaGfuUfc 216 GfsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd AfcUfcCfaGfcdTsdT C C TsdT  15GCUGGAGUU  16 GGCGGUCAC 210 GfscUfgGfaGfuUfc 222 GfsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG GfuGfaCfcGfcCfd AfcUfcCfaGfcdT C C T(invdT) (invdT) 15 GCUGGAGUU  16 GGCGGUCAC 211 gcUfgGfaGfuUfcG 223 dGsGfcGfgUfcAfcGfaCGUGACCGC GAACUCCAG fuGfaCfcGfcCfdT AfcUfcCfaGfcdT C C (invdT) (invdT) 15 GCUGGAGUU  16 GGCGGUCAC 204 GfcUfgGfaGfuUfc 224 pGfsGfcGfgUfcAfcGfCGUGACCGC GAACUCCAG GfuGfaCfcGfcCf aAfcUfcCfaGfc C C (invdT) (invdT)  15GCUGGAGUU  16 GGCGGUCAC  45 GCUGGAGUUCG  46 GGCGGUCACGAAC CGUGACCGCGAACUCCAG UGACCGCCdTdT UCCAGCdTdT C C  15 GCUGGAGUU  16 GGCGGUCAC 212GcuGGAGuucGuG 225 GGCGGUcACGAAC CGUGACCGC GAACUCCAG AccGccdTsdTUCcAGCdTsdT C C  17 CCACCCUGA  18 ACGCCGUAG  47 CCACCCUGACC  48ACGCCGUAGGUCA CCUACGGCG GUCAGGGUG UACGGCGUdTdT GGGUGGdTdT U G  17CCACCCUGA  18 ACGCCGUAG  77 CfcAfcCfcUfgAfc  78 paCfgCfcGfuAfgGfuCCCUACGGCG GUCAGGGUG CfuAfcGfgCfgUfd faGfgGfuGfgdTsdT U G TsdT  17CCACCCUGA  18 ACGCCGUAG 107 dCcdAcdCcdTgdA 108 padCgdCcdGudAgdGCCUACGGCG GUCAGGGUG cdCudAcdGgdCgd udCadGgdGudGgdTs U G TdTsdT dT  17CCACCCUGA  18 ACGCCGUAG 137 CfdCAfdCCfdCUf 138 pdACfdGCfdCGfdTACCUACGGCG GUCAGGGUG dGAfdCCfdTAfdC fdGGfdTCfdAGfdGG U G GfdGCfdGUfdTsdfdTGfdGdTsdT T  19 CGACUUCAA  20 GCCGUCCUCC  49 CGACUUCAAGG  50GCCGUCCUCCUUG GGAGGACGG UUGAAGUCG AGGACGGCdTdT AAGUCGdTdT C  19CGACUUCAA  20 GCCGUCCUCC  79 CfgAfcUfuCfaAfg  80 pgCfcGfuCfcUfcCfuUGGAGGACGG UUGAAGUCG GfaGfgAfcGfgCfd fgAfaGfuCfgdTsdT C TsdT  19CGACUUCAA  20 GCCGUCCUCC 109 dCgdAcdTudCadA 110 pgdCcdGudCcdTcdCuGGAGGACGG UUGAAGUCG gdGadGgdAcdGgd dTgdAadGudCgdTsd C CdTsdT T  19CGACUUCAA  20 GCCGUCCUCC 139 CfdGAfdCUfdTCf 140 pdGCfdCGfdTCfdCUGGAGGACGG UUGAAGUCG dAAfdGGfdAGfdG fdCCfdTUfdGAfdAGf C AfdCGfdGCfdTsddTCfdGdTsdT T  21 UUCAAGAUC  22 UGUUGUGGC  51 UUCAAGAUCCG  52UGUUGUGGCGGA CGCCACAAC GGAUCUUGA CCACAACAdTdT UCUUGAAdTdT A A  21UUCAAGAUC  22 UGUUGUGGC  81 UfuCfaAfgAfuCfc  82 puGfuUfgUfgGfcGfgCGCCACAAC GGAUCUUGA GfcCfaCfaAfcAfdT AfuCfuUfgAfadTsdT A A sdT  21UUCAAGAUC  22 UGUUGUGGC 111 dTudCadAgdAudC 112 pudGudTgdTgdGcdGCGCCACAAC GGAUCUUGA cdGcdCadCadAcd gdAudCudTgdAadTsd A A AdTsdT T  21UUCAAGAUC  22 UGUUGUGGC 141 UfdTCfdAAfdGAf 142 pdTGfdTUfdGUfdGGCGCCACAAC GGAUCUUGA dTCfdCGfdCCfdA fdCGfdGAfdTCfdTUf A A CfdAAfdCAfdTsddGAfdAdTsdT T  23 GGCAACUAC  24 CGCGGGUCU  53 GGCAACUACAA  54CGCGGGUCUUGUA AAGACCCGC UGUAGUUGC GACCCGCGdTdT GUUGCCdTdT G C  23GGCAACUAC  24 CGCGGGUCU  83 GfgCfaAfcUfaCfa  84 pcGfcGfgGfuCfuUfgAAGACCCGC UGUAGUUGC AfgAfcCfcGfcGfd UfaGfuUfgCfcdTsdT G C TsdT  23GGCAACUAC  24 CGCGGGUCU 113 dGgdCadAcdTadC 114 pcdGcdGgdGudCudTAAGACCCGC UGUAGUUGC adAgdAcdCcdGcd gdTadGudTgdCcdTsd G C GdTsdT T  23GGCAACUAC  24 CGCGGGUCU 143 GfdGCfdAAfdCUf 144 pdCGfdCGfdGGfdTCAAGACCCGC UGUAGUUGC dACfdAAfdGAfdC fdTUfdGUfdAGfdTUf G C CfdCGfdCGfdTsdTdGCfdCdTsdT  25 CCGGCAAGC  26 GGCACGGGC  55 CCGGCAAGCUG  56GGCACGGGCAGCU UGCCCGUGC AGCUUGCCG CCCGUGCCdTdT UGCCGGdTdT C G  25CCGGCAAGC  26 GGCACGGGC  85 CfcGfgCfaAfgCfu  86 pgGfcAfcGfgGfcAfgUGCCCGUGC AGCUUGCCG GfcCfcGfuGfcCfd CfuUfgCfcGfgdTsdT C G TsdT  25CCGGCAAGC  26 GGCACGGGC 115 dCcdGgdCadAgdC 116 pgdGcdAcdGgdGcdAUGCCCGUGC AGCUUGCCG udGcdCcdGudGcd gdCudTgdCcdGgdTsd C G CdTsdT T  25CCGGCAAGC  26 GGCACGGGC 145 CfdCGfdGCfdAAf 146 pdGGfdCAfdCGfdGGUGCCCGUGC AGCUUGCCG dGCfdTGfdCCfdC fdCAfdGCfdTUfdGCf C G GfdTGfdCCfdTsdTdCGfdGdTsdT  27 UGCCCAUCC  28 AGCUCGACC  57 UGCCCAUCCUG  58AGCUCGACCAGGA UGGUCGAGC AGGAUGGGC GUCGAGCUdTdT UGGGCAdTdT U A  27UGCCCAUCC  28 AGCUCGACC  87 UfgCfcCfaUfcCfu  88 paGfcUfcGfaCfcAfgGUGGUCGAGC AGGAUGGGC GfgUfcGfaGfcUfd faUfgGfgCfadTsdT U A TsdT  27UGCCCAUCC  28 AGCUCGACC 117 dTgdCcdCadTcdCu 118 padGcdTcdGadCcdAgUGGUCGAGC AGGAUGGGC dGgdTcdGadGcdT dGadTgdGgdCadTsdT U A dTsdT  27UGCCCAUCC  28 AGCUCGACC 147 UfdGCfdCCfdAUf 148 pdAGfdCUfdCGfdACUGGUCGAGC AGGAUGGGC dCCfdTGfdGUfdC fdCAfdGGfdAUfdGG U A GfdAGfdCUfdTsdfdGCfdAdTsdT T  29 CAAGUUCAG  30 GCCGGACAC  59 CAAGUUCAGCG  60GCCGGACACGCUG CGUGUCCGG GCUGAACUU UGUCCGGCdTdT AACUUGdTdT C G  29CAAGUUCAG  30 GCCGGACAC  89 CfaAfgUfuCfaGfc  90 pgCfcGfgAfcAfcGfcUCGUGUCCGG GCUGAACUU GfuGfuCfcGfgCfd fgAfaCfuUfgdTsdT C G TsdT  29CAAGUUCAG  30 GCCGGACAC 119 dCadAgdTudCadG 120 pgdCcdGgdAcdAcdGCGUGUCCGG GCUGAACUU cdGudGudCcdGgd cdTgdAadCudTgdTsd C G CdTsdT T  29CAAGUUCAG  30 GCCGGACAC 149 CfdAAfdGUfdTCf 150 pdGCfdCGfdGAfdCACGUGUCCGG GCUGAACUU dAGfdCGfdTGfdT fdCGfdCUfdGAfdAC C G CfdCGfdGCfdTsdTfdTUfdGdTsdT 151 GGAAUCUUA 152 UUGGAUCAA 153 GGAAUCuuAuAu 154uuGGAUcAAAuAuA UAUUUGAUC AUAUAAGAU uuGAUCcAsA AGAuUCcscsU CAA UCCCU 151GGAAUCUUA 152 UUGGAUCAA 265 (Chol)GGAAUCuu 154 uuGGAUcAAAuAuA UAUUUGAUCAUAUAAGAU AuAuuuGAUCcAs AGAuUCcscsU CAA UCCCU A 155 GGAUGAAGU 156ACUAAUCUC 157 GGAuGAAGuGG 158 ACuAAUCUCcACU GGAGAUUAG CACUUCAUCAGAuuAGudTsdT UcAUCCdTsdT U C 155 GGAUGAAGU 156 ACUAAUCUC 160(NH2C6)GfgAfuGf 159 pasCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCaAfgUfgGfaGfaUf CfuUfcAfuCfc(invdT) U C uAfgUf(invdT) 155 GGAUGAAGU 156ACUAAUCUC 226 gsgAfuGfaAfgUfg 266 pdAsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUfd aCfuUfcAfuCfcdTsdT U C TsdT 155 GGAUGAAGU 156 ACUAAUCUC227 gsgAfuGfaAfgUfg 266 pdAsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUf aCfuUfcAfuCfcdTsdT U C (invdT) 155 GGAUGAAGU 156ACUAAUCUC 228 GfsgAfuGfaAfgUf 267 pAfsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCgGfaGfaUfuAfgUf aCfuUfcAfuCfcdTsdT U C dTsdT 155 GGAUGAAGU 156 ACUAAUCUC229 GfsgAfuGfaAfgUf 268 pAfsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCgGfaGfaUfuAfgUf aCfuUfcAfuCfcdT U C dT(invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 230 ggAfuGfaAfgUfgG 238 dAsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCfaGfaUfuAfgUfdTs CfuUfcAfuCfcdTsdT U C dT 155 GGAUGAAGU 156 ACUAAUCUC231 GfsgAfuGfaAfgUf 267 pAfsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCgGfaGfaUfuAfgUf aCfuUfcAfuCfcdTsdT U C (invdT) 155 GGAUGAAGU 156ACUAAUCUC 232 ggAfuGfaAfgUfgG 238 dAsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCfaGfaUfuAfgUf CfuUfcAfuCfcdTsdT U C (invdT) 155 GGAUGAAGU 156 ACUAAUCUC233 GfgAfuGfaAfgUfg 239 AfsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUfd CfuUfcAfuCfcdT U C T(invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 234 GfgAfuGfaAfgUfg 240 AfsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUf CfuUfcAfuCfcdTsdT U C (invdT) 155 GGAUGAAGU 156 ACUAAUCUC231 GfsgAfuGfaAfgUf 241 pAfsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCgGfaGfaUfuAfgUf aCfuUfcAfuCfc U C (invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 234 GfgAfuGfaAfgUfg 159 pasCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUf CfuUfcAfuCfc(invdT) U C (invdT) 155 GGAUGAAGU 156ACUAAUCUC 235 gsgAfuGfaAfgUfg 242 pdAsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUfd aCfuUfcAfuCfcdT U C T(invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 236 GfgAfuGfaAfgUfg 240 AfsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUfd CfuUfcAfuCfcdTsdT U C TsdT 155 GGAUGAAGU 156 ACUAAUCUC227 gsgAfuGfaAfgUfg 243 pdAsCfuAfaUfcUfcCf GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUf aCfuUfcAfuCfc U C (invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 237 ggAfuGfaAfgUfgG 244 dAsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCfaGfaUfuAfgUfdT CfuUfcAfuCfcdT U C (invdT) (invdT) 155 GGAUGAAGU 156ACUAAUCUC 232 ggAfuGfaAfgUfgG 245 dAsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCfaGfaUfuAfgUf CfuUfcAfuCfc(invdT) U C (invdT) 155 GGAUGAAGU 156ACUAAUCUC 234 GfgAfuGfaAfgUfg 246 AfsCfuAfaUfcUfcCfa GGAGAUUAG CACUUCAUCGfaGfaUfuAfgUf CfuUfcAfuCfc(invdT) U C (invdT) 155 GGAUGAAGU 156ACUAAUCUC 157 GGAuGAAGuGG 158 ACuAAUCUCcACU GGAGAUUAG CACUUCAUCAGAuuAGudTsdT UcAUCCdTsdT U C 161 GCAAAGGCG 162 UGAGUUGGC 165(NH2C6)GfcAfaAf 166 puGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGgGfcGfuGfcCfaAfc GfcCfuUfuGfc(invdT) A C UfcAf(invdT) 161 GCAAAGGCG 162UGAGUUGGC 167 (NH2C6)GcAAAG 168 UGAGUUGGcACGC UGCCAACUC ACGCCUUUGGcGuGccAAcucAd CUUUGCdTsdT A C TsdT 161 GCAAAGGCG 162 UGAGUUGGC 179GalNAc-(NH2C6)- 166 puGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu GfcCfuUfuGfc(invdT) A C GfcCfaAfcUfcAf (invdT) 161GCAAAGGCG 162 UGAGUUGGC 180 GalNAc-(NH2C6)- 168 UGAGUUGGcACGC UGCCAACUCACGCCUUUG GcAAAGGcGuGcc CUUUGCdTsdT A C AAcucAdTsdT 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 185 pUfsGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 186 psdTdGaGfuUfgGfcA UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf fcGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 187 psdTsGfaGfuUfgGfcA UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf fcGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 188 pdTsdGaGfuUfgGfcA UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf fcGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 189 pdTsGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 190 pusGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 166 puGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 191 psUfGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 192 UfsGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 193 psUfsGfaGfuUfgGfcA UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf fcGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 194 psdTsdGaGfuUfgGfc UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf AfcGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 195 usGfaGfuUfgGfcAfcG UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf fcCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 162UGAGUUGGC 181 GfcAfaAfgGfcGfu 196 uGfaGfuUfgGfcAfcGf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 178 TGAGUUGGC181 GfcAfaAfgGfcGfu 197 dTsGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 198 psdTGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 178TGAGUUGGC 181 GfcAfaAfgGfcGfu 199 dTsdGaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161 GCAAAGGCG 162UGAGUUGGC 183 (Chol)GfcAfaAfgG 190 pusGfaGfuUfgGfcAfc UGCCAACUCACGCCUUUG fcGfuGfcCfaAfcUf GfcCfuUfuGfc(invdT) A C cAf(invdT) 161GCAAAGGCG 162 UGAGUUGGC 181 GfcAfaAfgGfcGfu 196 uGfaGfuUfgGfcAfcGfUGCCAACUC ACGCCUUUG GfcCfaAfcUfcAf cCfuUfuGfc(invdT) A C (invdT) 161GCAAAGGCG 162 UGAGUUGGC 181 GfcAfaAfgGfcGfu 195 usGfaGfuUfgGfcAfcGUGCCAACUC ACGCCUUUG GfcCfaAfcUfcAf fcCfuUfuGfc(invdT) A C (invdT) 161GCAAAGGCG 162 UGAGUUGGC 181 GfcAfaAfgGfcGfu 192 UfsGfaGfuUfgGfcAfcUGCCAACUC ACGCCUUUG GfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161GCAAAGGCG 178 TGAGUUGGC 181 GfcAfaAfgGfcGfu 197 dTsGfaGfuUfgGfcAfcUGCCAACUC ACGCCUUUG GfcCfaAfcUfcAf GfcCfuUfuGfc(invdT) A C (invdT) 161GCAAAGGCG 178 TGAGUUGGC 181 GfcAfaAfgGfcGfu 199 dTsdGaGfuUfgGfcAfUGCCAACUC ACGCCUUUG GfcCfaAfcUfcAf cGfcCfuUfuGfc A C (invdT) (invdT) 161GCAAAGGCG 162 UGAGUUGGC 182 GcAAAGGcGuGcc 168 UGAGUUGGcACGC UGCCAACUCACGCCUUUG AAcucAdTsdT CUUUGCdTsdT A C 161 GCAAAGGCG 162 UGAGUUGGC 184GfcAfaAfgGfcGfu 168 UGAGUUGGcACGC UGCCAACUC ACGCCUUUG GfcCfaAfcUfcACUUUGCdTsdT A C 161 GCAAAGGCG 162 UGAGUUGGC 179 GalNAc-(NH2C6)- 166puGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfuGfcCfuUfuGfc(invdT) A C GfcCfaAfcUfcAf (invdT) 161 GCAAAGGCG 162UGAGUUGGC 179 GalNAc-(NH2C6)- 190 pusGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu GfcCfuUfuGfc(invdT) A C GfcCfaAfcUfcAf (invdT) 161GCAAAGGCG 162 UGAGUUGGC 179 GalNAc-(NH2C6)- 185 pUfsGfaGfuUfgGfcAfUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu cGfcCfuUfuGfc A C GfcCfaAfcUfcAf(invdT) (invdT) 161 GCAAAGGCG 178 TGAGUUGGC 179 GalNAc-(NH2C6)- 263psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc AC GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 162 UGAGUUGGC 179GalNAc-(NH2C6)- 166 puGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu GfcCfuUfuGfc(invdT) A C GfcCfaAfcUfcAf (invdT) 161GCAAAGGCG 162 UGAGUUGGC 179 GalNAc-(NH2C6)- 190 pusGfaGfuUfgGfcAfcUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu GfcCfuUfuGfc(invdT) A CGfcCfaAfcUfcAf (invdT) 161 GCAAAGGCG 162 UGAGUUGGC 179 GalNAc-(NH2C6)-262 pUfsGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu cGfcCfuUfuGfcA C GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 256 TGAGUUGGC 179GalNAc-(NH2C6)- 263 psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu A C GfcCfaAfcUfcAf AfcGfcCfuUfuGfc (invdT) (invdT) 161GCAAAGGCG 162 UGAGUUGGC 257 Chol-(NH2C6)- 166 puGfaGfuUfgGfcAfcUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu GfcCfuUfuGfc(invdT) A CGfcCfaAfcUfcAf (invdT) 161 GCAAAGGCG 162 UGAGUUGGC 257 Chol-(NH2C6)- 190pusGfaGfuUfgGfcAfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfuGfcCfuUfuGfc(invdT) A C GfcCfaAfcUfcAf (invdT) 161 GCAAAGGCG 162UGAGUUGGC 257 Chol-(NH2C6)- 264 pUfsGfaGfuUfgGfcAf UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu cGfcCfuUfuGfc A C GfcCfaAfcUfcAf (invdT) (invdT) 161GCAAAGGCG 256 TGAGUUGGC 257 Chol-(NH2C6)- 263 psdTdGfaGfuUfgGfcUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf(invdT) (invdT) 161 GCAAAGGCG 162 UGAGUUGGC 258 Chol-(NH2C6)- 168UGAGUUGGcACGC UGCCAACUC ACGCCUUUG GcAAAGGcGuGcc CUUUGCdTsdT A CAAcucAdTsdT 161 GCAAAGGCG 256 TGAGUUGGC 257 Chol-(NH2C6)- 263psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc AC GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 256 TGAGUUGGC 259Chol-C6SSC6- 263 psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfuAfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 256TGAGUUGGC 260 Chol-AUC- 263 psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu AfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf (invdT) (invdT) 161GCAAAGGCG 256 TGAGUUGGC 261 Chol-Cathepsin- 263 psdTdGfaGfuUfgGfcUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf(invdT) (invdT) 161 GCAAAGGCG 256 TGAGUUGGC 257 Chol-(NH2C6)- 263psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc AC GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 256 TGAGUUGGC 259Chol-C6SSC6- 263 psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfuAfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf (invdT) (invdT) 161 GCAAAGGCG 256TGAGUUGGC 260 Chol-AUC- 263 psdTdGfaGfuUfgGfc UGCCAACUC ACGCCUUUGGfcAfaAfgGfcGfu AfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf (invdT) (invdT) 161GCAAAGGCG 256 TGAGUUGGC 261 Chol-Cathepsin- 263 psdTdGfaGfuUfgGfcUGCCAACUC ACGCCUUUG GfcAfaAfgGfcGfu AfcGfcCfuUfuGfc A C GfcCfaAfcUfcAf(invdT) (invdT) 163 GGAUCAUCU 164 GUAAGACUU 169 (NH2C6)GGAUfCf 170GUfAAGACfUfUfGA CAAGUCUUA GAGAUGAUC AUfCfUfCfAAGUf GAUfGAUfCfCfdTsd C CCfUfUfACfdTsdT T 171 UGACCACAG 172 UUUAAUCCG 173 (NH2C6)UfgAfcCf 174pusUfuAfaUfcCfgAfc UCGGAUUAA ACUGUGGUC aCfaGfuCfgGfaUfuUfgUfgGfuCfa(invdT) A A AfaAf(invdT) 171 UGACCACAG 172 UUUAAUCCG 175(NH2C6)uGAccAc 176 puUuAAUCCGACU UCGGAUUAA ACUGUGGUC AGucGGAuuAAAGUGGucAdTsdT A A dTsdT 171 UGACCACAG 172 UUUAAUCCG 175 (NH2C6)uGAccAc177 UUuAAUCCGACUG UCGGAUUAA ACUGUGGUC AGucGGAuuAAA UGGUcAdTsdT A A dTsdT247 GUUGGUGAA 248 CUGAGCUCC 251 (NH2C6)GfuUfgGf 252 pCfsUfgAfgCfuCfcAfUGGAGCUCA AUUCACCAA uGfaAfuGfgAfgCf uUfcAfcCfaAfc G C uCfaGf(invdT)(invdT) 247 GUUGGUGAA 248 CUGAGCUCC 251 (NH2C6)GfuUfgGf 255pCfsUfgAfgCfuCfcAf UGGAGCUCA AUUCACCAA uGfaAfuGfgAfgCfuUfcAfcCfaAfcdTsdT G C uCfaGf(invdT) 249 GGUCCUGUU 250 AUUCACCAA 253(NH2C6)GfgUfcCf 254 pAfsUfuCfaCfcAfaCf GUUGGUGAA CAACAGGACuGfuUfgUfuGfgUf aAfcAfgGfaCfcdTsdT U C gAfaUf(invdT)

What is claimed is:
 1. A compound of formula (I):

wherein Y is a linker group selected from —(CH₂)₃— or—C(O)—NH—(CH₂—CH₂—O)_(p)—CH₂—CH₂—; R¹ is —(C1-6) alkyl; —(CH₂)-naphthyl;or —(CH₂)_(m)-phenyl, which phenyl is unsubstituted or up to four timessubstituted with a substituent independently selected from —NO₂, —CN,Halogen, —O—(CH₂)-phenyl, —O—(C1-6) alkyl, or —C(O)—NH₂; R² is hydrogen;—(CH₂)_(k)—NH—C(Ph)₃, which phenyl rings are unsubstituted orindependently substituted with —O—(C1-4)alkyl; —(CH₂)_(k)—C(O)—NH₂;—(CH₂)_(k)-phenyl; —(C1-6) alkyl, which is unsubstituted or oncesubstituted with —S—CH₃; R³ is —NH-phenyl, which phenyl group is furthersubstituted with a substituent independently selected from —(CH₂)—OH; or—(CH₂)—O—C(O)—O-(4-nitro-phenyl); k is 1, 2, 3, 4, 5 or 6; m is 1, 2, 3or 4; n is 0 or 1; and p is an integer from 1 to
 20. 2. The compound ofclaim 1, having the conformation as shown in formula (Ia):


3. The compound of claim 1 wherein Y is —(CH₂)₃—.
 4. The compound ofclaim 3, wherein: Y is —(CH₂)₃—; R² is —(CH₂)_(k)—NH—C(Ph)₃, whichphenyl rings are unsubstituted or independently substituted with—O—(C1-4)alkyl; and R³ is —NH-phenyl, which phenyl group is furthersubstituted with —(CH₂)—O—C(O)—O-(4-nitro-phenyl); n is 0; and R¹ and kare as defined in claim
 1. 5. The compound of claim 1, wherein Y is—C(O)—NH—(CH₂—CH₂—O)_(p)—CH₂—CH₂—.
 6. The compound of claim 5, wherein:Y is —C(O)—NH—(CH₂—CH₂—O)_(p)—CH₂—CH₂—; R² is —(CH₂)_(k)—NH—C(Ph)₃,which phenyl rings are unsubstituted or independently substituted with—O—(C1-4)alkyl; and R³ is —NH-phenyl, which phenyl group is furthersubstituted with —(CH₂)—O—C(O)—O-(4-nitro-phenyl); n is 0; and R¹, k andp have the meanings given above.